Bendamustine derivatives and methods of using same

ABSTRACT

The present invention is directed to bendamustine esters and bendamustine amides and their use for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/184,968, filed on Jun. 16, 2016, which is a continuation of U.S.application Ser. No. 14/868,278, filed on Sep. 28, 2015, which is acontinuation of U.S. application Ser. No. 14/699,965, filed on Apr. 29,2015, which is a continuation of PCT Application No. PCT/US2013/069550,filed on Nov. 12, 2013, entitled “Bendamustine Derivatives and Methodsof Using Same,” which claims the benefit of U.S. Provisional ApplicationNo. 61/725,213, filed Nov. 12, 2012, and U.S. Provisional ApplicationNo. 61/776,951, filed Mar. 12, 2013, the entireties of which areincorporated by reference herein.

TECHNICAL FIELD

The present invention is directed to esters and amides of bendamustinefor use in treating cancer.

BACKGROUND

Bendamustine,4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid:

is marketed as the hydrochloride salt under the trade names RIBOMUSTINand TREANDA and is a compound that has been used successfully for thetreatment of blood cancers such as chronic lymphocytic leukemia,Hodgkin's disease, non-Hodgkin's lymphoma, and multiple myeloma. Theseproducts are administered as intravenous infusions.

Use of bendamustine for the treatment of solid tumors is limited,however, by the compound's chemical instability in aqueous environment.Indeed, bendamustine has been reported as having a half-life of onlyabout 6-10 minutes in vivo. As a result, circulating levels ofbendamustine are not sustained for a long enough time for bendamustineto reach tumors outside of the circulatory system. Methods forincreasing the circulation time of bendamustine are needed.

SUMMARY

The present invention is directed to compounds of formula I:

wherein R₁ is C₆-C₂₄alkyl or polyethylene glycol; or pharmaceuticallyacceptable salt forms thereof. Methods of using compounds of formula Ifor the treatment of solid and non-solid cancer tumors are alsodescribed.

The invention is also directed to the use of compounds of formula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol; or pharmaceuticallyacceptable salt forms thereof for the treatment of solid and non-solidcancer tumors.

The invention is further directed to compounds of formula II:

wherein R₂ is C₁-C₂₄alkylene; and R₃ is —COOC₁₋₃alkyl; or R₂-R₃ isC₁-C₂₄alkyl; or pharmaceutically acceptable salt forms thereof. Methodsof using compounds of formula II for the treatment of solid andnon-solid cancer tumors.

Nanoparticles including compounds of Formula I or IA, as well aslyophilized compositions comprising those nanoparticles, are also withinthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts plasma levels of certain embodiments of the invention inCD-1 mice, dosing at 3 mg/kg i.v. in 3% DMSO, 30% Solutol, 100 μL.

FIG. 2 depicts the effects of bendamustine hydrochloride and certainembodiments of the invention on tumor volumes of mice bearing MDA-MB-231xenografts.

FIG. 3 depicts the amount of bendamustine observed over time aftertreating MDA-MB-231 breast tumor S9 with certain embodiments of theinvention.

FIG. 4 depicts the amount of bendamustine observed over time aftertreating H460 non small cell lung tumor S9 with certain embodiments ofthe invention.

FIG. 5 depicts plasma levels of bendamustine in rats after dosing oneembodiment of the invention in rats using different formulations.

FIG. 6 depicts plasma levels of one embodiment of the invention in ratsafter dosing that embodiment in rats using different formulations.

FIG. 7 depicts Cryo-TEM images of nanoparticles of one embodiment of theinvention, bendamustine C₁₄ ester.

FIG. 8 depicts plasma levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 9 depicts blood levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 10 depicts brain levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 11 depicts liver levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 12 depicts lung levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 13 depicts spleen levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 14 depicts kidney levels of bendamustine and one embodiment of theinvention, bendamustine C₁₂ ester (as liquid and nanoparticleformulations), after dosing rats at 30 mg/kg i.v., 1 mL/kg withbendamustine C₁₂ ester.

FIG. 15 depicts plasma, blood, and organ levels of bendamustine in ratafter administration of one embodiment of the invention, bendamustineC₁₂ ester (as liquid formulation), after dosing rats at 30 mg/kg i.v., 1mL/kg.

FIG. 16 depicts plasma, blood, and organ levels of one embodiment of theinvention, bendamustine C₁₂ ester, in rat after dosing rats at 30 mg/kgi.v., 1 mL/kg with bendamustine C₁₂ ester (as liquid formulation).

FIG. 17 depicts plasma, blood, and organ levels of bendamustine in ratafter administration of one embodiment of the invention, bendamustineC₁₂ ester (as nanoparticle formulation), after dosing rats at 30 mg/kgi.v., 1 mL/kg.

FIG. 18 depicts plasma, blood, and organ levels of one embodiment of theinvention, bendamustine C₁₂ ester, in rat after dosing rats at 30 mg/kgi.v., 1 mL/kg with bendamustine C₁₂ ester (as nanoparticle formulation).

FIG. 19 depicts plasma levels of bendamustine in rat afteradministration of embodiments of the invention, bendamustine PEG-2000ester and bendamustine PEG-5000 ester, after dosing rats at 3 mg-eq/kgi.v., 1 mL/kg. Comparison is with TREANDA.

FIG. 20 depicts plasma levels of bendamustine in CD-1 mice after dosingembodiments of the invention at 3 mg/kg i.v., 3% DMSO, 30% Solutol.

FIG. 21 depicts a representative nanoparticle embodiment of theinvention at a magnification of 52,000×. Observed in the sample are:spherical particles that appear evenly denser than the surroundingbuffer (left-most arrows), small particles in the background (right-mostarrow). Insets show the two particles denoted by the left-most arrows ata larger scale. Distance between crosses in the left image is 28 nm,between crosses in the right inset is 43 nm. Scale Bar: 200 nm.

FIG. 22 depicts a representative nanoparticle embodiment of theinvention at a magnification of 52,000×. Observed in the sample are:spherical particles that appear evenly denser than the surroundingbuffer (left-most arrows), small particles in the background (right-mostarrow). Insets show the two particles denoted by the left-most arrows ata larger scale. Distance between crosses in the left image is 28 nm,between crosses in the right inset is 43 nm. Scale Bar: 200 nm.

FIG. 23 depicts tumor volumes following administration of VELCADE®,bendamustine, and bendamustine C12 ester nanoparticles.

FIG. 24 depicts body weight measurements following administration ofVELCADE®, bendamustine, and bendamustine C12 ester nanoparticles.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It has been discovered that converting the carboxylic acid moiety ofbendamustine to a C₁-C₂₄alkyl ester group, a polyethylene glycol estergroup, or a C₁-C₂₄alkyl amide group results in compounds that providelonger circulating times for bendamustine. While not wishing to be boundto any particular theory, it is presumed that the ester or amide moietyreduces the solubility of the bendamustine molecule, resulting in aprotective effect against the aqueous environment. Over time, the esteror amide moiety is hydrolyzed to reveal the carboxylic acid moiety ofthe active bendamustine molecule. The overall result is thatbendamustine is generated over time.

By varying the number of carbons in the ester/amide moiety, thelipophilicity of the resulting bendamustine derivative can be modified.Increasing lipophilicity has been correlated to increased stability ofthe ester/amide and longer circulating times of bendamustine.

Within the scope of the invention are compounds of formula IA:

wherein R is C₁-C₂₄alkyl or polyethylene glycol; or a pharmaceuticallyacceptable salt form thereof Compounds of formula IA are useful for thetreatment of solid or non-solid cancer tumors in patients.

Compounds of the invention can be formulated into pharmaceuticalcompositions comprising the compound of formula IA, or apharmaceutically acceptable salt form thereof, and a pharmaceuticallyacceptable carrier or diluent. In preferred pharmaceutical compositionsof the invention, R is C₁₀-C₂₄alkyl. Preferably, R is C₁₀alkyl. Alsopreferred is where R is C₁₀alkyl. Other preferred embodiments includethose where R is C₁₄alkyl. Compositions where R is C₁₆alkyl are alsopreferred.

Other embodiments of the invention include nanoparticles comprising acompound of formula IA.

Also within the scope of the invention are methods of treating solid ornon-solid cancer tumors in patients comprising administering to thepatient a compound of formula IA. Preferred solid or non-solid tumorsinclude chronic lymphocytic leukemia, Hodgkin's disease, indolentnon-Hodgkin's lymphoma (T-cell lymphoma, B-cell lymphoma), aggressivenon-Hodgkin's lymphoma, multiple myeloma, acute lymphocytic leukemia,breast cancer or lung cancer (small cell lung cancer (SCLC) andnon-small cell lung cancer (NSCLC), for example). Other solid andnon-solid cancer tumors are also envisioned as being treatable withcompounds and compositions of the invention, such as for example,sarcoma, bladder cancer, cervical cancer, testicular cancer, melanoma,glioblastoma, colon cancer, head and neck cancer, ovarian cancer, andprostate cancer. Other solid and non-solid cancer tumors are alsoenvisioned as being treatable with compounds of the invention, forexample, breast cancer, pancreatic cancer, and gastric cancer.

Preferred compounds of the invention are those of formula I:

wherein R₁ is C₆-C₂₄alkyl or polyethylene glycol; or a pharmaceuticallyacceptable salt form thereof. Compounds of formula I are useful for thetreatment of solid or non-solid cancer tumors in patients.

In preferred embodiments, R₁ is C₈-C₂₄alkyl. In other embodiments, R₁ isC₁₀-C₂₄alkyl. In yet other embodiments, R₁ is C₁₂-C₂₄alkyl. In stillother embodiments, R₁ is C₁₄-C₂₄alkyl. Also preferred are thosecompounds of formula I wherein R₁ is C₁₆-C₂₄alkyl. In other embodiments,R₁ is C₁₈-C₂₄alkyl.

In other embodiments, R₁ is C₁₀alkyl. In yet other embodiments, R₁ isC₁₂alkyl. In still other embodiments, R₁ is C₁₄alkyl. In otherembodiments, R₁ is C₁₆alkyl.

Also within the scope of the invention are pharmaceutical compositionscomprising a compound of formula I and a pharmaceutically acceptablecarrier or diluent.

Other embodiments of the invention include nanoparticles comprising acompound of formula I.

Also within the scope of the invention are methods of treating cancercomprising administering to a patient a compound of formula I. A numberof cancers, including those that involve solid tumors as well as thosethat do not involve solid tumors may be amenable to such treatment.These cancers include chronic lymphocytic leukemia, Hodgkin's disease,indolent non-Hodgkin's lymphoma (T-cell lymphoma, B-cell lymphoma),aggressive non-Hodgkin's lymphoma, multiple myeloma, acute lymphocyticleukemia, breast cancer or lung cancer (small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), for example). Additional cancersthat are also envisioned as being treatable with compounds andcompositions of the invention are those characterized by the presence ofsolid tumors, include sarcoma, bladder cancer, cervical cancer,testicular cancer, melanoma, glioblastoma, colon cancer, head and neckcancer, ovarian cancer, and prostate cancer. Other solid and non-solidcancer tumors are also envisioned as being treatable with compounds ofthe invention, for example, breast cancer, pancreatic cancer, andgastric cancer.

Particularly preferred compounds of the invention include:

Also within the scope of the invention are compounds of formula II:

wherein R₂ is C₁-C₂₄alkylene; and R₃ is —COOC₁₋₃alkyl; or R₂-R₃ isC₁-C₂₄alkyl; or a pharmaceutically acceptable salt form thereof.Compounds of formula II are useful for the treatment of solid ornon-solid cancer tumors in patients.

In preferred embodiments of compounds of formula II, R₂-R₃ isC₈-C₂₄alkyl. In other embodiments, R₂-R₃ is C₁₀-C₂₄alkyl. In still otherembodiments, R₂-R₃ is C₁₂-C₂₄alkyl. In yet other embodiments, R₂-R₃ isC₁₄-C₂₄alkyl. Also preferred is when R₂-R₃ is C₁₆-C₂₄alkyl. In otherembodiments, R₂-R₃ is C₁₈-C₂₄alkyl.

Preferably, for compounds of formula II, R₂-R₃ is C₁₀alkyl. Alsopreferred is when R₂-R₃ is C₁₀alkyl. In other embodiments, R₂-R₃ isC₁₄alkyl. In yet other embodiments, R₂-R₃ is C₁₀alkyl.

In other embodiments, R₂ is C₂alkylene and R₃ is —COOCH₃.

Preferred compounds of formula II include:

Also within the scope of the invention are pharmaceutical compositionscomprising a compound of formula II and a pharmaceutically acceptablecarrier or diluent.

Other embodiments of the invention include nanoparticles comprising acompound of formula II.

In one embodiment of the invention, the compounds and compositions ofthe invention are used to treat patients who are resistant to one ormore chemotherapeutic agents, such as, for example, alkylating agents.Exemplary alkylating agents to which patients may be resistant include:nitrogen mustards; ethylenimines; alkylsulfonates; triazenes;piperazines; and nitrosureas. More specific examples of the varioustypes of chemotherapeutic agents to which patients can become resistantare listed below. Patients resistant to one or more of these agentswould benefit by treatment with the compounds and compositions of theinvention.

Nitrogen Mustards

Mechlorethamine, marketed under the trade name Mustargen®, is given byinjection to treat Hodgkin's disease and non-Hodgkin's lymphoma, and asa palliative therapy for breast and lung cancers, and given as a topicaltreatment for skin lesions of mycosis fungoides (cutaneous T-celllymphoma).

Ifosfamide, sold under the trade name Ifex®, is used to treat bothHodgkin's and non-Hodgkin's lymphoma, as well as recurrent testicularcancer and germ cell tumors, sarcomas, lung cancer, bladder cancer, headand neck cancer, and cervical cancer.

Melphalan is a chemotherapy drug sold under the brand name Alkeran®, andis also referred to as L-PAM or phenylalanine mustard. It is used totreat multiple myeloma, ovarian cancer, neuroblastoma, rhabdomyosarcoma,and breast cancer.

Chlorambucil is sold by the trade name Leukeran®, and is most widelyused to treat chronic lymphocytic leukemia, malignant lymphomasincluding lymphosarcoma, giant follicular lymphoma, and Hodgkin'sdisease. It has also been successfully used to treat non-Hodgkin'slymphoma, breast, ovarian and testicular cancer, Waldenstrom'smacroglobulinemia, thrombocythemia, and choriocarcinoma.

Cyclophosphamide is marketed as Cytoxan® or Neosar®, and is used totreat Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, chroniclymphocytic leukemia, chronic myelocytic leukemia, acute myelocyticleukemia, acute lymphocytic leukemia, t-cell lymphoma, multiple myeloma,neuroblastoma, retinoblastoma, rhabdomyosarcoma, Ewing's sarcoma;breast, testicular, endometrial, ovarian, and lung cancers.

Nitrosoureas

Streptozocin is sold under the trade name Zanosar®, and is used to treatislet cell pancreatic cancer.

Carmustine is also known as BiCNU® or BCNU, and is used for some kindsof brain tumors, glioblastoma, brainstem glioma, medulloblastoma,astrocytoma, ependymoma, and metastatic brain tumors. It is also used intreatment for multiple myeloma, Hodgkin's disease, non-Hodgkin'slymphoma, melanoma, lung cancer, and colon cancer.

Lomustine, also known as CCNU or CeeNU®, is used to treat primary andmetastatic brain tumors, Hodgkin's disease and non-Hodgkin's lymphoma,and has also been used for melanoma, lung, and colon cancer.

Alkyl Sulfonates

Busulfan, sold under trade names Busulfex® and Myleran®, is used totreat chronic myelogenous leukemia.

Triazines

Dacarbazine is sold under the trade name DTIC-Dome® and is used to treatmetastatic malignant melanoma, Hodgkin's disease, soft tissue sarcomas,neuroblastoma, fibrosarcomas, rhabdomyosarcoma, islet cell carcinoma,and medullary thyroid carcinoma.

Temozolomide is sold under the trade name Temodar®, and is used to treatthe specific types of brain tumors anaplastic astrocytoma andglioblastoma multiforme.

Ethylenimines

Thiotepa, known under the trade name Thioplex®, is an alkylating agentused to treat breast cancer, ovarian cancer, Hodgkin's disease, andnon-Hodgkin's lymphoma.

Altretamine is sold under the trade name Hexalen®, and is also calledhexamethylmelamine or HMM. It is used to treat ovarian cancer.

As used herein, “C₁-C₂₄alkyl” refers to straight or branched, saturatedhydrocarbon groups containing from one to 24 carbon atoms.Representative alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, n-dodecyl, etc. Within the scopeof the invention, “C₁-C₂₄alkyl” also encompasses “cycloalkyl,” whichrefers to monocyclic, bicyclic, and tricyclic saturated hydrocarbons,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclobutyl,bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptyl, adamantyl, and the like.

As used herein “polyethylene glycol,” also referred to as “PEG,” refersto polymers of the general formula H(OCH₂CH₂)_(n)O— orH(OCH₂CH₂)_(n)OCH₃, wherein n is at least 4. The preferred PEG has anaverage molecular weight of from about 200 to about 5000 Daltons, with amore preferred PEG from about 2000 to about 5000 Daltons.

As used herein, “pharmaceutically acceptable carrier or diluent” refersto solvents, dispersion media, coatings, bulking agents, stabilizingagents, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanol,sugars such as trehalose and sucrose, polyalcohols such as mannitol,sorbitol, mixtures of sugars and polyalcohols, and sodium chloride.Pharmaceutically acceptable carriers may further include auxiliarysubstances such as wetting or emulsifying agents, preservatives, andbuffers.

As used herein, “pharmaceutical composition” refers to a compositionsuitable for administration in medical or veterinary use. Such compoundswill preferably include a compound of the invention in combination withone or more carriers and/or diluents. Such compositions are alsoreferred to as “formulations.”

As used herein, “administering” refers to any means within the art bywhich compounds of the invention can be delivered to the patient.Preferred administration methods include local administration, that is,administration of the compounds of the invention directly to thelocation where the effect of the compounds is desired, and systemicadministration. Examples of administration methods include, but are notlimited to, oral, enteric, sublingual, sublavial, subcutaneous, nasal,intravenous, intraarterial, intramuscular, and intraperitonealadministration.

As used herein, “solid tumor” refers to a malignant tumor that is alocalized mass of tissue. Examples of solid cancer tumors includelymphomas, sarcomas, and carcinomas and include breast cancer, braincancer, bone cancer, colon cancer, pancreatic cancer, lung cancer, andthe like.

As used herein, a “non-solid tumor cancer” refers most commonly tohematologic cancers, that is, malignant cancers of the blood. Examplesof non-solid tumor cancers include chronic lymphocytic leukemia,Hodgkin's disease, indolent non-Hodgkin's lymphoma (T-cell lymphoma,B-cell lymphoma), multiple myeloma, and the like.

As used herein, “nanoparticles” refers to a particle having an averagediameter of about 0.2 μm or less, preferably about 0.1 μm or less, asmeasured by Malvern Zetasizer.

Experimental Section Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid methyl ester (bendamustine C₁ ester)

Method A: To a 1 L three neck, round bottom flask equipped with anoverhead stirrer, condenser with nitrogen sweep, and thermocouple withtemperature controller was charged4-(5-amino-1-methyl-1H-benzoimidazol-2-yl)-butyric acid methyl ester(10.2 g, 41.2 mmol. 1.0 eq), and chloroacetic acid (81.9 g, 866 mmol),and 20 mL of dry tetrahydrofuran (THF). The slurry was stirred in a tapwater bath to allow all of the solids to be dissolved. Borane-THF (288mL, 288 mmol) was added slowly via an additional funnel over 25 minutes.When the addition of BH₃-THF was complete, the resulting reactionsolution was stirred at room temperature for 1.5 hours and then heatedat 58° C. using a heat mantle for 45 minutes. The reaction was cooledand held at room temperature overnight and then quenched with methanol(10 mL). The resulting solution was concentrated to approximatelyone-third weight by evaporation on the rotary evaporator and neutralizedto pH 8-9 with an aqueous solution of sodium hydroxide in an ice-waterbath. The solid was collected by vacuum filtration, washed with water(200 mL), then reslurried with a dilute aqueous solution of sodiumbicarbonate (50 mL) for 20 minutes. Filtration was followed by dryingwith house vacuum at room temperature overnight, giving a tan solid (9.6g, 63% yield, 93A % purity) ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8Hz, 1H), 6.92 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 3.70 (brs, 8H), 3.66 (s, 3H), 3.59 (s, 3H), 2.83 (t, J=7.4 Hz, 2H), 2.48 (t,J=7.4 Hz, 2H, overlapped partially with DMSO), 2.01 (quint, J=7.4Hz,2H); LC/MS (ESI, m/z) 372 (M+1), mp 60-63° C. dec.

Method B: To a 2 L three-neck glass vessel equipped with a heatingmantle, thermocouple, condenser, nitrogen inlet/outlet, and overheadstirrer was charged bendamustine HCl (50.0 g, 126.7 mmol, 1.0 eq.),methanol (500 mL), and methanesulfonic acid (2.47 mL, 38.1 mmol). Thereaction mixture was heated to reflux and stirred at 65° C. for onehour. The reaction solution was cooled to 40° C. and concentrated undervacuum. Water (500 mL) was added to the concentrated residue, and asaturated aqueous solution of NaHCO₃ (150 mL) was used to neutralize themixture to pH 6 over 1.5 hours. The product was collected by filtration,washed with water (150 mL) and dried at 40° C. under vacuum, giving awhite, powdery solid, 44.2 g (94% yield) with 98.4A % purity by HPLC.

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid ethyl ester (bendamustine C₂ ester)

Method A: To a 1 L three-neck glass vessel equipped with a heatingmantle, thermocouple, condenser, nitrogen inlet/outlet, and overheadstirrer was charged bendamustine HCl (30.0 g, 76 mmol, 1.0 eq.), ethanol(300 mL), and methanesulfonic acid (1.48 mL, 22.8 mmol). The reactionmixture was heated at 70° C. for one hour. The reaction solution wascooled to 40° C. and concentrated under vacuum. Water (300 mL) was addedto the concentrated residue, and a saturated aqueous solution of NaHCO₃(115 mL) was used to neutralize the mixture to pH 6 over 1.5 hours. Theproduct was collected by filtration, washed with water (100 mL) anddried at 40° C. under vacuum, giving a white solid, 28.6 g (97% yield)with 99.2A % purity by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8Hz, 1H), 6.92 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 4.04(quint, J=7.12 Hz, 2H), 3.70 (br s, 8H), 3.66 (s, 3H), 2.83 (t, J=7.4Hz, 2 H), 2.45 (t, J=7.4 Hz, 2H, overlapped partially with DMSO), 2.00(quint, J=7.4Hz, 2H), 1.18 (t, J=7.12 Hz, 3H).

Method B: To a 500 mL three-neck glass flask equipped with a heatingmantle, thermocouple, condenser, nitrogen inlet/outlet, and overheadstirrer was charged 4-(5-amino-1-methyl-1H-benimidazol-2-yl)-butyricacid ethyl ester (6.4 g, 1.0 eq.), chloroacetic acid (42.5 g), andtetrahydrofuran (THF, 13 mL). The resulting mixture was stirred for 1.5hours in a water bath at room temperature. Borane-THF (150 mL) was addedover 20 minutes. Once the charge was complete, the reaction mixture washeated to 55-58° C. and stirred for 1.5 hours. In-process analysis byHPLC showed 94A % of the desired product. The reaction was cooled toroom temperature and telescoped to the next step of hydrolysis togenerate bendamustine.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid butyl ester (bendamustine C₄ ester): A 250 mL three neck roundbottom flask was equipped with an overhead stirrer, thermocouple,temperature controller and nitrogen sweep then charged with 10.0 g(25.34 mmol) of bendamustine hydrochloride, 1.9 g (2.35 mL, 25.6 mmol,1.01 eq) of 1-butanol, 5.3 g (25.6 mmol, 1.01 eq) ofdicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol, 0.1eq) of DMAP. The reaction was stirred at room temperature overnight atwhich time an in process analysis indicated the reaction was complete.Solids were removed by vacuum filtration and washed with 25 mL of MDC.The filtrate was washed with saturated aqueous sodium bicarbonatesolution (2×100 mL), DI water (1×100 mL) and brine (1×100 mL) beforedrying over sodium sulfate, filtering and concentrating to dryness invacuo to a brown oil. The oil was triturated with 25 mL of MDC and thesolid impurities were removed by vacuum filtration and washed with 25 mLof MDC. The filtrate was concentrated to dryness in vacuo to yield 9.5 g(22.8 mmol, 90%) of the product as a clear light brown oil with an HPLCpurity of 94.5A %. ¹H NMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H),7.08 (d, J=2.32 Hz, 1H), 6.77 (dd, J=2.36, 8.8 Hz, 1H), 4.05 (t, J=6.76Hz, 2 H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz,2H), 2.49 (t, J=7.1 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 2H),0.89 (t, J=4.56 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid hexyl ester (bendamustine C₆ ester)

Method A: A 250 mL three neck round bottom flask was equipped with anoverhead stirrer, thermocouple, temperature controller and nitrogensweep then charged with 10.0 g (25.34 mmol) of bendamustinehydrochloride, 2.62 g (3.22 mL, 25.6 mmol, 1.01 eq) of 1-hexanol, 5.3 g(25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of MDCand 0.31 g (2.54 mmol, 0.1 eq) of DMAP. The reaction was stirred at roomtemperature overnight at which time an in process analysis indicated thereaction was complete. Solids were removed by vacuum filtration andwashed with 25 mL of MDC. The filtrate was washed with saturated aqueoussodium bicarbonate solution (2×100 mL), DI water (1×100 mL) and brine(1×100 mL) before drying over sodium sulfate, filtering andconcentrating to dryness in vacuo to a brown oil. The oil was trituratedwith 25 mL of MDC and the solid impurities were removed by vacuumfiltration and washed with 25 mL of MDC. The filtrate was concentratedto dryness in vacuo to yield 8.91 g (20.1 mmol, 79%) of the product as aclear light brown oil with an HPLC purity of 91.9A %. ¹H NMR (400 MHz,CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H), 7.08 (d, J=2.32 Hz, 1H), 6.77 (dd,J=2.36, 8.8 Hz, 1H), 4.05 (t, J=6.76 Hz, 2 H), 3.72 (m, 4H), 3.69 (s,3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz, 2H), 2.49 (t, J=7.1 Hz, 2H), 2.18(m, 2H), 1.60 (m, 2H), 1.32 (m, 6H), 0.89 (t, J=4.56 Hz, 3H).

Method B: A one liter 4-necked round bottom flask equipped with anoverhead stirrer, thermocouple and nitrogen in/oulet was charged with 30g (76.0 mmol) of bendamustine hydrochloride and 300 mL ofdichloromethane. Agitation was begun and 10.6 mL (7.69 g, 76.0 mmol) oftriethylamine was added via syringe then stirred for 15 minutes at roomtemperature before adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDAC, 21.86 g, 114 mmol) and n-hexyl alcohol (9.57 mL, 7.84 g, 76.8mmol). The cloudy white reaction mixture became a clear solution afterstirring for 20 minutes. Agitation was continued for 22.5 h at roomtemperature and 30° C. for one hour until reaction was complete by HPLCanalysis. DI water (300 mL) was charged to quench the reaction and thepH was adjusted to 6 using 1N HCl. The layers were separated, aqueouswas extracted with dichloromethane (100 mL), before combining theorganic phases and drying over sodium sulfate. After filtration andconcentration to dryness under vacuum a clear yellow oil was obtainedwhich was purified by column chromatography (25 to 50% ethyl acetate inheptane). A total of 16.5 g (37.3 mmol, 49.1%) was recovered as a thickyellow oil with 99.1 A % purity by HPLC.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyric aidoctyl ester (bendamustine C₈ ester): A 250 mL three neck round bottomflask was equipped with an overhead stirrer, thermocouple, temperaturecontroller and nitrogen sweep then charged with 10.0 g (25.34 mmol) ofbendamustine hydrochloride, 3.33 g (4.03 mL, 25.6 mmol, 1.01 eq) of1-octanol, 5.3 g (25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC),100 mL of MDC and 0.31 g (2.54 mmol, 0.1 eq) of DMAP. The reaction wasstirred at room temperature overnight at which time an in processanalysis indicated the reaction was complete. Solids were removed byvacuum filtration and washed with 25 mL of MDC. The filtrate was washedwith saturated aqueous sodium bicarbonate solution (2×100 mL), DI water(1×100 mL) and brine (1×100 mL) before drying over sodium sulfate,filtering and concentrating to dryness in vacuo to a brown oil. The oilwas triturated with 25 mL of MDC and the solid impurities were removedby vacuum filtration and washed with 5 mL of MDC. The filtrate wasconcentrated to dryness in vacuo to yield 9.7 g (20.5 mmol, 81%) of theproduct as a clear light brown oil with an HPLC purity of 91.9A %. ¹HNMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H), 7.08 (d, J=2.28 Hz, 1H),6.77 (dd, J=2.4, 8.76 Hz, 1H), 4.05 (t, J=6.8 Hz, 2 H), 3.72 (m, 4H),3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.44 Hz, 2H), 2.49 (t, J=7.12 Hz,2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 10H), 0.89 (t, J=6.72 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid decyl ester (bendamustine C₁₀ ester)

Method A: A 250 mL three necked round bottom flask equipped with a stirbar, thermocouple and nitrogen in/outlet was charged with 10.0 g (25.3mmol) of bendamustine hydrochloride, 4.9 mL (4.08 g, 25.6 mmol, 1.01 eq)of decyl alcohol, 5.3 g (25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of dichloromethane and 0.31 g (2.53 mmol, 0.1eq) of N,N-dimethylamino pyridine (DMAP). The reaction mixture wasstirred at room temperature for 18 hours at which time an HPLC analysisindicated the reaction was complete. Solids were removed by vacuumfiltration and the filtrate was washed with DI water (2×100 mL) andsaturated sodium bicarbonate (1×100 mL) before being dried over sodiumsulfate. The organic phase was filtered to remove the drying agent thenconcentrated to dryness in vacuo to give 9.6 g (19.2 mmol, 75.9%) of thedesired product as a low melting white solid with an HPLC purity of94.1A %. ¹H NMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H), 7.08 (d,J=2.32 Hz, 1H), 6.77 (dd, J=2.4, 8.76 Hz, 1H), 4.05 (t, J=6.76 Hz, 2 H),3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz, 2H), 2.49(t, J=7.08 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 14H), 0.87 (t,J=6.68 Hz, 3H).

Method B: A 250 mL 4-necked round bottom flask equipped with a magneticstir bar, thermocouple and nitrogen in/oulet was charged with 10 g (25.3mmol) of bendamustine hydrochloride and 100 mL of dichloromethane.Agitation was begun and 3.53 mL (2.56 g, 25.3 mmol) of triethylamine wasadded via syringe then stirred for 15 minutes at room temperature beforeadding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 7.28 g, 38mmol) and n-decyl alcohol (4.88 mL, 4.04 g, 25.5 mmol). The cloudy whitereaction mixture became a clear solution after stirring for 20 minutes.Agitation was continued for 20 h at room temperature until reaction wascomplete by HPLC analysis. DI water (100 mL) was charged to quench thereaction and the pH was adjusted to 6-7 using 1N HCl. The layers wereseparated, aqueous was extracted with dichloromethane (25 mL), beforecombining the organic phases and drying over sodium sulfate. Afterfiltration and concentration to dryness under vacuum a clear yellow oilwas obtained which was purified by column chromatography (20 to 60%ethyl acetate in heptane). A total of 11.2 g (22.42 mmol, 88.6%) wasrecovered as a thick yellow oil with 98.9 A % purity by HPLC.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyric aiddodecyl ester (bendamustine C₁₂ ester)

Method A: A 250 mL three neck round bottom flask was equipped with anoverhead stirrer, thermocouple, temperature controller and nitrogensweep then charged with 10.0 g (25.34 mmol) of bendamustinehydrochloride, 4.77 g (25.6 mmol, 1.01 eq) of 1-dodecanol, 5.3 g (25.6mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31g (2.54 mmol, 0.1 eq) of DMAP. The reaction was stirred at roomtemperature overnight at which time an in process analysis indicated thereaction was complete. Solids were removed by vacuum filtration andwashed with 25 mL of MDC. The filtrate was washed with saturated aqueoussodium bicarbonate solution (2×100 mL), DI water (1×100 mL) and brine(1×100 mL) before drying over sodium sulfate, filtering andconcentrating to dryness in vacuo to an off-white semi-solid. This solidwas triturated with 25 mL of MDC and the solid impurities were removedby vacuum filtration and washed with 5 mL of MDC. The filtrate wasconcentrated to dryness in vacuo to yield 11.53 g (21.9 mmol, 86.4%) ofthe product as an off-white semisolid with an HPLC purity of 93.7A %.

Method B: A 20 liter jacketed cylindrical ChemGlass reaction vesselequipped with a thermocouple, heater/chiller, nitrogen inlet/outlet,addition funnel, and vacuum line was charged with the free base ofbendamustine (374 g, 1.04 mol, 1.0 eq.),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 300 g, 1.57 mol,1.5 equivalents), and dichloromethane (DCM, 3.74 L, 10 volumes). Whilestirring 1-dodecanol (292.1 g, 1.57 mol, 1.5 equivalents) was added. Thereaction mixture was heated to then stirred at 27° C. for 4 hours. Thebatch was cooled and held at 20° C. overnight. The batch was then washedwith 3.75 L of water and the layers were separated. The aqueous portionwas re-extracted with 1.2 L of dichloromethane, and the combineddichloromethane portions were dried over Na₂SO₄. After filtering toremove the drying agent, the filtrate was concentrated in vacuo toproduce the product as a crude oil. Another batch of this reaction with374 g of free base of bendamustine under the same conditions was carriedout. The crude products from the two batches were combined, mixed with4494 mL of heptanes and heated to 45-50° C. The resultant solution wasallowed to slowly cool to room temperature, precipitating an off-whitesolid. The slurry was stirred overnight at room temperature and thesolid was isolated at 10° C. by vacuum filtration. The wet cake waswashed with 1 L of heptanes and reslurried with 2.5 L of heptane at20-22° C. overnight. The product was collected by filtration and washedtwice with 500 mL of heptanes each time. Drying the wet cakes overnightat 20-22° C. yielded 653 g (59% yield) of white solids at 99.0 A % byHPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8 Hz, 1H), 6.92 (d, J=2.3Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 3.99 (t, J=6.64 Hz, 2H), 3.70 (brs, 8H), 3.65 (s, 3H), 2.83 (t, J=7.4 Hz, 2 H), 2.45 (t, J=7.4 Hz, 2H,overlapped partially with DMSO), 2.01 (quint, J=7.4Hz, 2H), 1.54 (quint,J=6.9 Hz, 2H), 1.24 (m, 18H), 0.85 (t, J=6.8 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid tetradecyl ester (bendamustine C₁₄ ester)

Method A: A 500 mL three necked round bottom flask equipped with a stirbar, thermocouple and nitrogen in/outlet was charged with 10.0 g (25.3mmol) of bendamustine hydrochloride, 6.5 g (30.4 mmol, 1.2 eq) oftetradecyl alcohol, 6.3 g (30.4 mmol, 1.2 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of dichloromethane and 0.62 g (5.1 mmol, 0.2eq) of N,N-dimethylamino pyridine (DMAP). The reaction mixture wasstirred at room temperature for 20 hours at which time an HPLC analysisindicated the reaction was complete. Solids were removed by vacuumfiltration and the wetcake was washed with 50 mL of dichloromethanebefore concentrating the filtrate to dryness in vacuo. The resultantlight orange oil was triturated with 50 mL of dichloromethane and theundesired solids were removed by vacuum filtration. The filtrate wasonce again concentrated to dryness in vacuo to yield 16.5 g of asemi-solid which was shown by ¹H NMR to contain tetradecanol and DMAP.The residue was chromatographed on 150 g of silica gel 60, 230-400 mesheluting with 1500 mL of MDC, 1000 mL of 0.5% methanol/MDC and 2000 mL of1% methanol/MDC collecting 100-150 mL fractions. Fractions containingthe desired product were combined and concentrated to dryness in vacuo.The residue was again triturated with 30 mL of MDC and the undesiredsolids removed by filtration. The filtrate was concentrated in vacuo toyield 5.0 g (9.2 mmol, 36.4%) of the desired product as a light purplesolid with a purity of 95.0A %.

Method B: To a 150 mL three-neck glass vessel equipped with athermocouple, condenser, nitrogen inlet/outlet, and overhead mechanicalstirrer was charged the free base of bendamustine (16.0 g, 44.6 mmol,1.0 eq.), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 9.42 g,49.2 mol), 1-tetradecanol (10.6 g, 49.2 mmol) and dichloromethane (120mL). The reaction mixture was stirred at 27° C. overnight. The reactionsolution was cooled to room temperature and washed with 100 mL of water.While stirring, 1M aqueous HCl solution was added to adjust the pH ofthe aqueous layer to pH 3-4. The layers were separated. The aqueousportion was re-extracted with 100 mL of dichloromethane, and thecombined dichloromethane portions were dried over MgSO₄. After filteringto remove the drying agent, the filtrate was concentrated in vacuo toproduce the product as a waxy yellow solid. The solid was slurried in 80mL of heptanes at room temperature overnight. The product was collectedby filtration and dried, giving a white solid, 20.6 g (83.4% yield) with97.2A % purity by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.8 Hz,1H), 6.91 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.3 Hz, 1H), 3.98 (t,J=6.64 Hz, 2H), 3.70 (br s, 8H), 3.66 (s, 3H), 2.83 (t, J=7.4 Hz,2 H),2.45 (t, J=7.4 Hz, 2H, overlapped partially with DMSO), 2.01 (quint,J=7.3 Hz, 2H), 1.54 (m, 2 H), 1.23 (m, 18 H), 0.85 (t, J=7.12 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid pentadecyl ester (bendamustine C₁₅ ester): A 250 mL three neckround bottom flask was equipped with an overhead stirrer, thermocouple,temperature controller and nitrogen sweep then charged with 10.0 g(25.34 mmol) of bendamustine hydrochloride, 5.85 g (25.6 mmol, 1.01 eq)of pentadecanol, 5.3 g (25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide(DCC), 100 mL of MDC and 0.31 g (2.54 mmol, 0.1 eq) of DMAP. Thereaction was stirred at room temperature overnight at which time an inprocess analysis indicated the reaction was complete. Solids wereremoved by vacuum filtration and washed with 25 mL of MDC. The filtratewas washed with saturated aqueous sodium bicarbonate solution (2×100mL), DI water (1×100 mL) and brine (1×100 mL) before drying over sodiumsulfate, filtering and concentrating to dryness in vacuo to an off-whitesolid. This solid was triturated with 25 mL of MDC and the solidimpurities were removed by vacuum filtration and washed with 5 mL ofMDC. The filtrate was concentrated to dryness in vacuo to yield 10.8 g(19.0 mmol, 75%) of the product as an off-white solid with an HPLCpurity of 94.6A %. ¹H NMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H),7.08 (d, J=2.32 Hz, 1H), 6.78 (dd, J=2.4, 8.76 Hz, 1H), 4.05 (t, J=6.8Hz, 2 H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz,2H), 2.49 (t, J=7.08 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 24H),0.88 (t, J=6.68 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid hexadecyl ester (bendamustine C₁₆ ester): A 250 mL three neck roundbottom flask was equipped with an overhead stirrer, thermocouple,temperature controller and nitrogen sweep then charged with 10.0 g(25.34 mmol) of bendamustine hydrochloride, 6.2 g (25.6 mmol, 1.01 eq)of hexadecanol, 5.3 g (25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide(DCC), 100 mL of MDC and 0.31 g (2.54 mmol, 0.1 eq) of DMAP. Thereaction was stirred at room temperature overnight at which time an inprocess analysis indicated the reaction was complete. Solids wereremoved by vacuum filtration and washed with 25 mL of MDC. The filtratewas washed with saturated aqueous sodium bicarbonate solution (2×100mL), DI water (1×100 mL) and brine (1×100 mL) before drying over sodiumsulfate, filtering and concentrating to dryness in vacuo to an off-whitesolid. This solid was triturated with 25 mL of MDC and the solidimpurities were removed by vacuum filtration and washed with 5 mL ofMDC. The filtrate was concentrated to dryness in vacuo to yield 13.1 g(22.5 mmol, 88.8%) of the product as an off-white solid with an HPLCpurity of 94.0A %.¹H NMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.76 Hz, 1H),7.08 (d, J=2.32 Hz, 1H), 6.78 (dd, J=2.36, 8.72 Hz, 1H), 4.05 (t, J=6.8Hz, 2 H), 3.72 (m, 4H), 3.69 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.4 Hz,2H), 2.49 (t, J=7.08 Hz, 2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 26H),0.88 (t, J=6.68 Hz, 3H).

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid Oleoyl ester (bendamustine C₁₈ ester)

Method A: To a 250 mL, three neck, round bottom flask equipped with anoverhead stirrer, condenser with nitrogen sweep, heating mantle withtemperature controller and thermocouple was charged 1-octadecanol (50 g,185 mmol, 7.3 eq). The solid was heated to melt it before adding slowly4-(5-amino-1-methyl-1H-benzoimidazol-2-yl)-butyric acid (10 g, 25.3mmol. 1.0 eq) and sulfuric acid (0.5 mL). The resulting slurry wasstirred at 70° C. for 6 hours and then cooled to 56° C., where methylenechloride (150 mL) was added. The reaction mixture was cooled to roomtemperature and washed with water (100 mL). After phase separation,another extraction was performed with methylene chloride (100 mL). Theorganic phases were combined and dried over MgSO₄. The drying agent wasremoved by filtration. The filtrate was concentrated and subjected toSFC isolation. A white solid was obtained from evaporation of solvent inthe SFC fractions under reduced pressure and dried with house vacuum atroom temperature for 5 days, giving 1.2 g of the desired product in 7.1%yield and with 95.4A % purity. ¹H NMR (400 MHz, CDCl3) δ 7.18 (d, J=8.8Hz, 1H), 7.09 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.8, 2.4 Hz, 1H), 4.05 (t,J=6.8 Hz, 2H), 3.74 (m, 7H), 3.62 (m, 4H), 2.93 (t, J=7.4 Hz, 2H), 2.49(t, J=7.1 Hz, 2H), 2.22 (quint, J=7.1 Hz, 2H), 1.60 (quint, J=7.1 Hz,2H), 1.28 (m, 30H), 0.88 (t, J=7.1 Hz, 3H); LC/MS (ESI, m/z) 610(M+1).

Method B: To a 500 mL, three neck, round bottom flask equipped with astir bar, nitrogen sweep, and thermocouple was charged with bendamustineHCl (5.04 g, 12.8 mmol), 1-octadecanol (4.15 g, 15.3mmol),N,N′-Dicyclohexylcarbodiimide (DCC, 3.17 g, 15.4 mmol),4-Dimethylaminopyridine (DMAP, 0.31 g, 2.56 mmol) and methylene chloride(250 mL). The resulting slurry was stirred at room temperature for 16hours. A solid was produced and removed from the reaction mixture byfiltration. The filtrate was washed with water (150 mL). After phaseseparation, the organic phase dried over MgSO₄. The drying agent wasremoved by filtration and the filtrate was concentrated and subjected toISCO chromatographic purification with a mixture of EtOAc and heptanes,giving a white solid 5.68g (73% yield) with 99A % purity.

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid docosyl ester (bendamustine C₂₂ ester): A 250 mL three necked roundbottom flask equipped with a stir bar, thermocouple and nitrogenin/outlet was charged with 5.0 g (12.7 mmol) of bendamustinehydrochloride, 4.2 g (12.9 mmol, 1.01 eq) of docosyl alcohol, 2.7 g(12.9 mmol, 1.01 eq) of dicyclohexyl carbodiimide (DCC), 50 mL ofdichloromethane (MDC) and 0.16 g (1.27 mmol, 0.1 eq) ofN,N-dimethylamino pyridine (DMAP). The reaction mixture was stirred atroom temperature overnight at which time an HPLC analysis indicated thereaction was complete. Solids were removed by vacuum filtration and thewetcake was washed with 50 mL. Two alternate purification procedureswere developed. The filtrate was washed with DI water (2×150 mL), driedover sodium sulfate, filtered and concentrated to dryness in vacuo. Theresultant waxy residue was chromatographed on 80 g of silica gel 60,230-400 mesh eluting with a gradient beginning with 100% MDC, then 0.5%methanol/MDC and finally 1% methanol/MDC collecting 100-150 mLfractions. Fractions containing the desired product were combined andconcentrated to dryness in vacuo. The residue was again triturated with20 mL of MDC and the undesired solids removed by filtration. Thefiltrate was concentrated in vacuo to yield 3.65 g (5.5 mmol, 43.1%) ofthe desired product as a waxy white solid with a purity of 95.7A %. ¹HNMR (400 MHz, CDCl₃) δ 7.17 (d, J=8.72 Hz, 1H), 7.08 (d, J=2.28 Hz, 1H),6.78 (dd, J=2.36, 8.72 Hz, 1H), 4.05 (t, J=6.76 Hz, 2 H), 3.72 (m, 4H),3.70 (s, 3H), 3.63 (m, 4H), 2.91 (t, J=7.44 Hz, 2H), 2.49 (t, J=7.08 Hz,2H), 2.18 (m, 2H), 1.60 (m, 2H), 1.32 (m, 38H), 0.88 (t, J=6.64 Hz, 3H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid 2-dodecyl ester (branched bendamustine C₁₂ ester): A 250 mL threeneck round bottom flask was equipped with an overhead stirrer,thermocouple, temperature controller and nitrogen sweep then chargedwith 10.0 g (25.34 mmol) of bendamustine hydrochloride, 4.77 g (5.75 mL,25.6 mmol, 1.01 eq) of 2-dodecanol, 5.3 g (25.6 mmol, 1.01 eq) ofdicyclohexylcarbodiimide (DCC), 100 mL of MDC and 0.31 g (2.54 mmol, 0.1eq) of DMAP. The reaction was stirred at room temperature overnight atwhich time an in process analysis indicated the reaction was complete.Solids were removed by vacuum filtration and washed with 25 mL of MDC.The filtrate was diluted with 200 mL of MDC then washed with 4% aqueoussodium bicarbonate solution (1×500 mL) before drying over sodiumsulfate, filtering and concentrating to dryness in vacuo to an off-whitesolid. This solid was triturated with 25 mL of MDC and the solidimpurities were removed by vacuum filtration and washed with 5 mL ofMDC. The filtrate was concentrated to dryness in vacuo to yield thecrude product which was shown to contain residual 2-dodecanol by ¹H NMR.The crude product was chromatographed using 100 g of silica gel 60,230-400 mesh, eluting with first 1 L of heptanes, then 500 mL of 3:1heptane/EtOAc, 500 of 2:1 heptane/EtOAc and finally 500 mL of 1:1heptane/EtOAc collecting 100 mL fractions. Product containing fractionswere combined and concentrated to dryness in vacuo to yield 5.35 g(10.16 mmol, 40%) of the product as a light purple viscous oil with anHPLC purity of 99.5A %. ¹H NMR (400 MHz, DMSO-d₆) δ 7.31 (d, J=8.76 Hz,1H), 6.93 (d, J=2.28 Hz, 1H), 6.78 (dd, J=2.36, 8.76 Hz, 1H), 4.8 (m,1H), 3.7 (s, 8H), 3.65 (s, 3H), 2.82 (t, J=7.4 Hz, 2H), 2.42 (t, J=7.36Hz, 2H), 2.00 (m, 2H), 1.50 (m, 2H), 1.25 (s, b, 16H), 1.14 (d, J=6.24,2H), 0.84 (t, J=6.68 Hz, 3H).

Preparation of Bendamustine C₁₂ Ester

A 20 liter jacketed cylindrical ChemGlass reaction vessel equipped withthermocouple, heater/chiller, nitrogen inlet, addition funnel,condenser, and vacuum line was charged with a slurry of 428 g (1.10mmol) of pretreated bendamustine hydrochloride in 10 volumes of trace GCanalysis grade methylene chloride. Agitation was set at 100 RPM and thejacket was set at 20° C. To this mixture was added diisopropylethylamine(213 ml, 1.1 eq) via an addition funnel over 10 minutes. After a 34minute hold, melted dodecanol (227 g, 1.1 eq) was added in one portion.After an 11 minute hold, EDCI (320.3 g, 1.5 eq) was added to the batch.The resulting clear yellow solution was agitated for 23.5 hours at ˜20°C. At this point, an IPC indicated 0.54% starting starting materialremained. Ten volumes of water were added and the reaction was agitatedfor an additional 15 minutes. The lower organic layer was drained,filtered through a 5 micron filter cartridge, and the filter cartridgewas rinsed with 1 volume of GC analysis grade methylene chloride. Themethylene chloride solution was concentrated in vacuo to afford thecrude product as a viscous yellow oil. Five volumes of filteredn-heptane were added to the oil and the mixture was concentrated invacuo to remove residual methylene chloride to yield 615 g of crudesolids with in 92.9 wt % translating to a 98.0% yield. Final purity was98.4% on a dry basis.

Purification of Bendamustine C₁₂ Ester

Crude CEP-40125 (1100 g API, 1250 g crude) was taken up in n-heptane (6volumes) and transferred to a 20 liter jacketed cylindrical ChemGlassreaction vessel equipped with thermocouple, heater/chiller, nitrogeninlet, condenser, and vacuum line. The slurry was warmed to 40° C. todissolve all solids. Upon reaching 32.6° C., dissolution occurred. Thereaction mixture was then cooled to 17.7° C. over 2.5 hours, at whichpoint the product precipitated. The reaction mixture was then re-warmedto 23° C. to dissolve fine particles over 26 minutes, and cooled to 4°C. over 2 hours. The solids were filtered through a sealed filter andwashed with 2 volumes of cold n-heptane over 4.5 hours. An IPC indicated0.20% residual dodecanol. The solids were dried under vacuum for 24hours at 30° C. to constant weight to afford 1018 g CEP-40125 in 98.3%purity, representing a 92.5% yield.

Preparation of4-{5-[Bis-(chloroethy)-amino]-1-methyl-1H-benzimidazol-2-yl}butyric acid5-decyl ester (branched bendamustine C₁₀ ester): A 250 mL three neckround bottom flask was equipped with an overhead stirrer, thermocouple,temperature controller and nitrogen sweep then charged with 3.0 g (7.6mmol) of bendamustine hydrochloride, 1.21 g (7.7 mmol, 1.01 eq) of5-decanol, 1.59 g (7.7 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC),30 mL of 1,2-ethylene dichloride (EDC) and 0.1 g (0.76 mmol, 0.1 eq) ofDMAP. The reaction was stirred at 75° C. for five days until an inprocess analysis indicated the reaction was complete. Solids wereremoved by vacuum filtration and washed with 5 mL of EDC. The filtratewas washed with 4% aqueous sodium bicarbonate solution (1×50 mL) beforedrying over sodium sulfate, filtering and concentrating to dryness invacuo. The residue was combined with the residue from a 10 g batch runcarried out under the same conditions and the combined batches werechromatographed. The chromatography was carried out using 100 g ofsilica gel 60, 230-400 mesh, eluting with first 2 L of heptanes, then 1L of 3:1 heptane/EtOAc and 1 L of 2:1 heptane/EtOAc collecting 100 mLfractions. Product containing fractions were combined and concentratedto dryness in vacuo to yield 3.97 g (7.96 mmol, 24.2%) of the product asa clear yellow oil with an HPLC purity of 99.4A %. ¹H NMR (400 MHz,DMSO-d₆) δ 7.31 (d, J=8.76 Hz, 1H), 6.93 (d, J=2.28 Hz, 1H), 6.78 (dd,J=2.40, 8.80 Hz, 1H), 4.8 (m, 1H), 3.7 (s, 8H), 3.65 (s, 3H), 2.83 (t,J=7.4 Hz, 2H), 2.44 (t, J=7.36 Hz, 2H), 2.02 (m, 2H), 1.48 (m, 4H), 1.25(s, b, 10H), 0.84 (m, 6H).

Preparation of4-{5-[Bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid cyclohexyl ester: A 250 mL three neck round bottom flask wasequipped with an overhead stirrer, thermocouple, temperature controllerand nitrogen sweep then charged with 10 g (25.34 mmol) of bendamustinehydrochloride, 2.56 g (2.7 mL, 25.6 mmol, 1.01 eq) of cyclohexanol, 5.3g (25.6 mmol, 1.01 eq) of dicyclohexylcarbodiimide (DCC), 100 mL of MDCand 0.31 g (2.54 mmol, 0.1 eq) of DMAP. The reaction slurry was stirredat RT for 18 h until an in process analysis indicated the reaction wascomplete. Two new major product peaks were observed. Solids were removedby vacuum filtration and washed with 5 mL of MDC. The filtrate waswashed with 4% aqueous sodium bicarbonate solution (2×100 mL) beforedrying over sodium sulfate, filtering and concentrating to dryness invacuo to yield a yellow oil with some solids present. ¹H NMR analysisindicated residual DMAP and cyclohexanol was along with DCC by-productswere present in addition to the desired product. The residue wasslurried with 50 mL of MDC to remove residual cyclohexanol thenconcentrated in vacuo and chromatographed. The chromatography wascarried out using 50 g of silica gel 60, 230-400 mesh, eluting with 1:1heptane/EtOAc collecting 100 mL fractions. Product containing fractionswere combined and concentrated to dryness in vacuo to yield 3.11 g (7.06mmol, 27.9%) of the product as an off-white solid with an HPLC purity of97.8A %. ¹H NMR (400 MHz, DMSO-d₆) δ 7.31 (d, J=8.76 Hz, 1H), 6.93 (d,J=2.28 Hz, 1H), 6.77 (dd, J=2.40, 8.80 Hz, 1H), 4.65 (m, 1H), 3.7 (s,8H), 3.65 (s, 3H), 2.83 (t, J=7.4 Hz, 2H), 2.44 (t, J=7.36 Hz, 2H), 2.00(m, 2H), 1.76 (m, 4H), 1.65 (m, 2H), 1.33 (m, b, 6H).

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid PEG-2000 ester (bendamustine PEG-2000 ester): To a 100 mLthree-neck glass vessel equipped with a stir bar, thermocouple,condenser, and nitrogen inlet/outlet was charged bendamustinehydrochloride (2.0 g, 5.1 mmol, 1.0 eq.) and dichloromethane (30 mL).Triethyl amine (0.71 mL, 5.1 mmol) was added to the slurry at 22° C. andstirred for 20 minutes. Methoxypolyethylene glycol 2000 (PEG-OMe-2000,12.2 g, 6.1 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDAC, 1.5 g, 7.6 mol) were added. The reaction mixture was stirred at22° C. for 5.5 hours, at this point addition of PEG-OMe-2000 (1.0 g) wasfollowed by stirring for 3 days through weekend. Water (20 mL) was addedand pH was adjusted to pH 5-6 by adding 1M hydrochloric acid. The phasesseparated slowly. The aqueous portion was re-extracted with 20 mL ofdichloromethane, and the combined dichloromethane portions were driedover MgSO₄. After filtering to remove the drying reagent, the filtratewas concentrated in vacuo to produce the product as a waxy solid. Thesolid was slurried in 10 mL of heptanes at room temperature. The productwas collected by filtration and dried at 30° C. under vacuum, giving awhite and powdery solid, 11.7 g (99% yield) with 97.9A % purity by HPLC.¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.6 Hz, 1H), 6.92 (d, J=2.2 Hz,1H), 6.78 (dd, J=8.8,2.2 Hz, 1H), 4.12 (t, J=4.8 Hz, 2H), 3.70 (m, 12H),3.60 (m, 3H), 3.51 (m, 224H), 3.43 (m, 4 H), 3.32 (m, 58 H), 2.84 (t,J=7.4 Hz,2 H), 2.45 (2H, overlapped partially with DMSO), 2.01 (quint,J=7.3 Hz, 2H).

Preparation of4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyricacid PEG-5000 ester (bendamustine PEG-5000 ester): To a 50 mL three-neckglass vessel equipped with a stir bar, thermocouple, condenser, andnitrogen inlet/outlet was charged bendamustine hydrochloride (0.5 g,1.27 mmol, 1.0 eq.) and dichloromethane (15 mL). Triethyl amine (0.18mL, 1.28 mmol) was added to the slurry at 22° C. and stirred for 20minutes along with methoxypolyethylene glycol 5000 (PEG-OMe-5000, 6.33g, 1.27 mmol) and, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC,0.36 g, 1.88 mol). The reaction mixture was stirred at 22° C. overnight.Water (20 mL) was added and pH was adjusted to pH 3-4 by adding 1Mhydrochloric acid. The phases separated slowly. The aqueous portion wasre-extracted with 10 mL of dichloromethane. The combined dichloromethaneportions were washed with brine (20 mL) and dried over MgSO₄. Afterfiltering to remove the drying agent, the filtrate was concentrated invacuo to produce the product as a waxy solid. The solid was slurried in20 mL of heptanes at room temperature. The product was collected byfiltration and dried at 30° C. under vacuum, giving a white and powderysolid, 5.24 g (77% yield) with 99A % purity by HPLC. ¹H NMR (400 MHz,DMSO-d6) δ 7.38 (d, J=12 Hz, 1H), 6.91 (d, J=2.4 Hz, 1H), 6.82 (d, J=10Hz, 1H), 4.12 (t, J=4.7 Hz, 2H), 3.72 (br s, 8H), 3.68 (m, 5H), 3.59 (m,3H), 3.51 (m, 436 H), 3.42 (m, 3H), 3.31 (m, 28 H), 2.88 (t, J=7.4 Hz,2H), 2.45 (t, 2H, overlapped partially with DMSO), 2.01 (quint, J=7.3 Hz,2H).

General Procedure for Transesterification of Bendamustine Methyl Ester

To a 50 mL three-neck glass vessel equipped with a stir bar,thermocouple, condenser with a Dean-Stark trap, and nitrogeninlet/outlet was charged bendamustine methyl ester (0.5 g, 1.27 mmol,1.0 eq.), catalyst (0.05-1.4 eq.), an appropriate solvent (5-15volumes), and an excess of dodecanol (5-10 eq.). The resulting reactionmixture was heated to reflux and monitored by HPLC. The results undervaried conditions are summarized below.

Dean- Start Catalyst Solvent Alcohol Temp Stark Time material ProductCatalyst (eq) Solvent Volumes Equivs (° C.) Trap (h) (HPLC A %) (HPLC A%) CH₃SO₃H 1.40 NA NA 2.50 30 N 3 3.8 94.5 CH₃SO₃H 1.40 DCM 10 2.50 30 N47 22.0 76.2 CH₃SO₃H 1.40 Toluene 10 2.50 70 N 24 19.3 69.7 I₂ 0.25Toluene 5 2.50 75 N 7 96.0 0.2 TiO(acac)₂ 0.05 Xylenes 10 1.01 130 N 792.7 4.0 TiO(acac)₂ 0.10 Xylenes 5 10.0 130 N 6 10.5 85.5 TiO(acac)₂0.10 Xylenes 5 10.0 160 N 5 9.0 78.6 TiO(acac)₂ 0.10 Xylenes 15 5.00 160Y 6 1.0 97.0 H₂SO₄ 0.5 Toluene 10 5.0 100 N 23 78.9 13.5 DMAP 0.5Toluene 10 5.0 100 N 23 83 0 p-TSA 0.5 Xylenes 10 5.0 130 N 21 67.2 15.6Sm(Oi-Pr)₃ 0.5 THF 10 5.0 25-45 N 27 2.6 90.5 Superbase 0.5 THF 10 5.025-45 N 27 3.3 49.8 Sm(Oi-Pr)₃ 0.1 Xylenes 15 5.0 160 N 8 72.8 24.3Sm(Oi-Pr)₃ 0.5 THF 10 5.0 40 N 16 95.8 2.5 TiO(acac)₂ 0.05 Xylenes 155.0 150 Y 3 0.3 96.9 TiO(acac)₂ 0.05 Toluene 15 5.0 105 N 70 78.9 15.9TiO(acac)₂ 0.10 Toluene 15 5.0 115 Y 22 ND 96.4 TiO(acac)₂ 0.05 Toluene15 5.0 130 N 24 83.2 4.1 TiO(acac)₂ 0.05 Toluene 15 5.0 115 Y 21 0.497.7

Preparation of Bendamustine Hydrochloride Amides

4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-N-decyl-butylamine(bendamustine C₁₀ amide): A 250 mL three neck round bottom flaskequipped with a stir bar, thermocouple, cooling bath, 60 mL pressureequalizing dropping funnel and nitrogen in/outlet was charged with 10.0g (25.3 mmol) of bendamustine hydrochloride, 10.6 g (27.8 mmol) of HATUand 100 mL of N,N-dimethylformamide (DMF). To this stirred yellowsolution was added 4.41 mL (3.27 g, 25.3 mmol) ofN,N-diisopropylethylamine (DIPEA). An exotherm to 27.1° C. was noted andthe solution became a darker yellow. The reaction was cooled to 6.6° C.where a solution of 6.2 mL (4.59 g, 35.5 mmol) of DIPEA, 5.11 mL (4.1 g,25.6 mmol) of decyl amine in 20 mL of DMF was added drop-wise over 13min at 2.7-7.6° C. Once addition was complete the reaction was allowedto stir at <10° C. for 1.5 hours at which time an in process analysisindicated the reaction was complete. The batch was quenched onto 200 mLof DI water and extracted with ethyl acetate (2×175 mL). The organicphases were combined, washed with 10% sodium hydrogen phosphate (1×200mL), 8% aqueous sodium bicarbonate (1×200 mL) and brine (1×200 mL)before concentrating to dryness in vacuo to give a sticky white solid.This solid was triturated with heptanes (75 mL) and became a flowablesolid which was isolated by vacuum filtration. The wetcake was dried ina vacuum oven at 25° C. overnight to yield 13.33 g (25.3 mmol, 100%) ofthe desired product as a white solid with an HPLC purity of 98.01A %. ¹HNMR (400 MHz, DMSO-d6) δ 7.72 (s, b, 1H), 7.33 (d, J=8.76 Hz, 1H), 6.91(d, J=2.28 Hz, 1H), 6.80 (dd, J=2.36, 8.8 Hz), 3.7 (s, 8H), 3.66 (s,3H), 3.01 (q, J=6.8, 12.68, 2H), 2.79 (t, J=7.44 Hz, 2H), 2.18 (t,J=7.36 Hz, 2H), 1.95 (m, 2H), 1.36 (m, 2H), 1.22 (s, b, 14), 0.84 (t,J=6.68 Hz, 3H).

4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-N-tetradecyl-butryamide(bendamustine C₁₄ amide): A 250 mL three neck round bottom flaskequipped with a stir bar, thermocouple, cooling bath, 60 mL pressureequalizing dropping funnel and nitrogen in/outlet was charged with 10.0g (25.3 mmol) of bendamustine hydrochloride, 10.6 g (27.8 mmol) of HATUand 100 mL of N,N-dimethylformamide (DMF). To this stirred yellowsolution was added 4.41 mL (3.27 g, 25.3 mmol) ofN,N-diisopropylethylamine (DIPEA). An exotherm to 27.1° C. was noted andthe solution became a darker yellow. The reaction was cooled to 3.3° C.where a solution of 6.2 mL (4.59 g, 35.5 mmol) of DIPEA, 5.75 gf (25.6mmol) of tetradecyl amine in 40 mL of DMF was added drop-wise over 6 minat <10° C. Once addition was complete the reaction became very thick anddifficult to stir. It was transferred to a 500 mL three neck roundbottom flask equipped with an overhead stirrer and thermocouple, thenstirred at RT for three hours at which time an in process analysisindicated the reaction was complete. The batch was quenched onto 400 mLof DI water and extracted with ethyl acetate (2×300 mL), the organicphases were combined, washed with 10% sodium hydrogen phosphate (1×300mL), 8% aqueous sodium bicarbonate (1×300 mL) and brine (1×300 mL)before drying over sodium sulfate, filtering and concentrating todryness in vacuo. The residue was purified by chromatography using 100 gof silica gel 60, 230-400 mesh, eluting with 1% MeOH/MDC (2 L), 2.5%MeOH/MDC (1 L) and 5% MeOH/MDC (1 L) collecting ˜100 mL fractions. Theproduct containing fractions were combined and concentrated to drynessin vacuo to yield 7.86 g (14.6 mmol, 57.6%) of the desired product as awhite solid with an HPLC purity of 97.3A %. ¹H NMR (400 MHz, DMSO-d6) δ7.72 (s, b, 1H), 7.33 (d, J=8.84 Hz, 1H), 6.91 (d, J=2.22 Hz, 1H), 6.80(dd, J=2.36, 8.84 Hz), 3.71 (s, 8H), 3.70 (s, 3H), 3.01 (q, J=6.8,12.68, 2H), 2.79 (t, J=7.44 Hz, 2H), 2.18 (t, J=7.36 Hz, 2H), 1.97 (m,2H), 1.36 (m, 2H), 1.28 (s, b, 22), 0.84 (t, J=7.04 Hz, 3H).

4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-N-octadecyl-butyramide(bendamustine C₁₈ amide): A 250 mL three neck round bottom flaskequipped with a stir bar, thermocouple, cooling bath, 60 mL pressureequalizing dropping funnel and nitrogen in/outlet was charged with 10.0g (25.3 mmol) of bendamustine hydrochloride, 10.6 g (27.8 mmol) of HATUand 100 mL of N,N-dimethylformamide (DMF). To this stirred yellowsolution was added 4.41 mL (3.27 g, 25.3 mmol) ofN,N-diisopropylethylamine (DIPEA). An exotherm to 27.1° C. was noted andthe solution became a darker yellow. The reaction was cooled to 2.0° C.where a suspension of 6.2 mL (4.59 g, 35.5 mmol) of DIPEA, 7.65 g (25.6mmol) of octadecyl amine in 0 mL of DMF was added via pipette. Onceaddition was complete the reaction became very thick and difficult tostir. It was warmed to room temperature and the magnetic stir bar wasreplaced with an overhead stirrer. The batch was stirred at RT overnightafter which time an in process analysis indicated the reaction wascomplete. The batch was quenched onto 300 mL of DI water and extractedwith dichloromethane (2×150 mL). The organic phases were combined,washed with 10% sodium hydrogen phosphate (1×300 mL), 8% aqueous sodiumbicarbonate (1×300 mL) and brine (1×300 mL) before drying over sodiumsulfate, filtering and concentrating to dryness in vacuo. The residuewas purified by chromatography using 100 g of silica gel 60, 230-400mesh, eluting with 1% MeOH/MDC (2 L), 2.5% MeOH/MDC (1 L) and 5%MeOH/MDC (1 L) collecting ˜100 mL fractions. The product containingfractions were combined and concentrated to dryness in vacuo to yield5.11 g (8.38 mmol, 33%) of the desired product as a white solid with anHPLC purity of 90.9A %. The major impurity was shown to be the C-16amide which results from an impurity in the starting amine. ¹H NMR (400MHz, DMSO-d6) δ 7.72 (s, b, 1H), 7.33 (d, J=8.84 Hz, 1H), 6.91 (d,J=2.22 Hz, 1H), 6.80 (dd, J=2.36, 8.84 Hz), 3.71 (s, 8H), 3.70 (s, 3H),3.01 (q, J=6.8, 12.68, 2H), 2.79 (t, J=7.44 Hz, 2H), 2.18 (t, J=7.36 Hz,2H), 1.97 (m, 2H), 1.36 (m, 2H), 1.28 (s, b, 30H), 0.85 (t, J=6.32 Hz,3H).

(S)-2-(4-{5-[Bis-(2-chloro-ethyl)-amino]-1-methyl-1H-benzoimidazol-2-yl}-butyrylamino)-propionicacid methyl ester: A 250 mL three neck round bottom flask equipped witha stir bar, thermocouple, cooling bath, addition funnel and nitrogenin/outlet was charged with 10 g (25.3 mmol) of bendamustinehydrochloride, 10.6 g (27.8 mmol) of HATU and 100 mL of DMF. The batchwas cooled to 1.9° C. where 8.8 mL (6.54 g, 50.6 mmol) of DIPEA wasadded over 2 minutes. The reaction exothermed to 9° C. and becameorange. A solution of 3.57 g (25.6 mmol) of L-alanine methyl esterhydrochloride and 6.2 mL (4.57 g, 35.4 mmol) of DIPEA in 20 mL of DMFwas added drop-wise over 10 minutes at 4.9-5.7° C. The reaction wasslowly warmed to RT over one hour and stirred for three hours at whichtime an in process assay indicated the reaction was complete. The batchwas quenched onto 400 mL of 1:1 ethyl acetate/DI water. The layers wereseparated, the organic was washed with 10% sodium hydrogen phosphate(1×200 mL), 8% sodium bicarbonate 91×200 mL) and brine (1×200 mL),before drying over sodium sulfate, filtering and evaporating to drynessin vacuo. The residue was purified by chromatography using 100 g ofsilica gel 60, 230-400 mesh, eluting with 1% MeOH/MDC (3 L), 2.5%MeOH/MDC (1 L) and 5% MeOH/MDC (500 mL) collecting ˜100 mL fractions.The product containing fractions were combined and concentrated todryness in vacuo to yield 7.1 g (16.0 mmol, 63.3%) of the desiredproduct as a white solid with an HPLC purity of 97.4A %. ¹H NMR (400MHz, DMSO-d6) δ 8.25 (d, J=6.92 Hz), 1H), 7.40 (d, J=8.84 Hz, 1H), 6.92(d, J=2.2 Hz, 1H), 6.86 (dd, J=2.32, 8.88 Hz), 4.25 (q, 1H), 3.72 (s,8H), 3.71 (s, 3H), 3.62 (s, 3H), 2.87 (t, J=7.48 Hz, 2H), 2.25 (t,J=7.52 Hz, 2H), 1.99 (m, 2H), 1.26 (d, J=7.32 Hz, 3H).

General Procedure for Preparing Solution Formulations of BendamustineEsters of the Invention:

A stock solution of the bendamustine ester of the invention wasdissolved in a 60/40 (v/v) mixture of dimethylacetamide (“DMA”) andSolutol® HS-15 at about 100 mg/mL concentration. The mixture was stirredat room temperature until dissolved. The resulting stock solution, whichwas stable for several months, was diluted with 0.9% saline to thedesired concentration and dosed within about 2 hours.

Bendamustine C₁₄ Ester Solution Formulation: A stock solution wasprepared by dissolving 320.1 mg of bendamustine C₁₄ ester in 4 mL of a60/40 (v/v) mixture of DMA and Solutol® HS-15. The mixture was stirredfor about 2 hours until dissolved. Prior to dosing, the stock solutionwas diluted by removing 1.00 mL of the stock solution and adding 16.20mL saline and stirring for 5 minutes at room temperature. The resultingformulation was 3 mg-equ/mL bendamustine.

Pretreatment to Remove Residual Ethanol:

Bendamustine hydrochloride (277 g) was charged into a 5 L evaporationflask followed by 2 volumes of DI water and 5 volumes of acetone. Thesolvents were distilled under vacuum at a maximum temperature of 40° C.over 5.5 hours. An additional 5 volumes of acetone were added and theresulting slurry was rotated at atmospheric pressure on the rotaryevaporator at 35° C. for 15 minutes. The batch was then filtered througha sealed sintered glass funnel and the resulting white solids wererinsed with 2 volumes of acetone. The solids were transferred to adrying tray and dried at 40° C. to constant weight for 17 hours beforeisolating 266 g (96.0%) of product in 99.8A % purity with KF of 0.26%.

General Procedure for Preparing Human Serum Albumin (“HSA”) NanoparticleFormulations of Bendamustine Esters of the Invention

Nanoparticles were formed from an O/W emulsion using dichloromethane andHSA as a surfactant. The oil phase was prepared by dissolving thedesired amount of bendamustine ester in dichloromethane at aconcentration of about 120 mg/mL. The water phase was prepared bydissolving 2-4× the amount of HSA (w/w base on the bendamustine ester)in 5-15× the volume of water (w/w based on dichloromethane). Typically,mannitol was added to the aqueous phase at 5-10% to make the solutionisotonic for injection and to provide a pharmaceutically appropriateproduct post lyophilization. The O/W emulsion was formed by emulsifyingthe oil phase and the water phase using and IKA hand-held homogenizer atmedium intensity for about 30 seconds. The nanoparticles were formed byprocessing the crude O/W emulsion through a Microfluidizer® highpressure homogenizer (5 passes at about 30,000 psi) to provide 50-100 nmsized particles, as measured using dynamic light scattering (MalvernZetasizer). The solvent was removed under vacuum and the resultingconcentrate was either lyophilized or stored frozen prior to dosing.These formulations exhibited good physical and chemical stability.

The lyophilized nanoparticles were reconstituted and analyzed usingcryo-Transmission Electron Microscopy (c-TEM). The majority of thenanoparticles were 20-40 nm solid spheres that were readily dispersed inwater. A minority of particles were in the 125 nm range. The particlesall had a smooth surface.

Bendamustine C₁₂ Ester HSA Nanoparticles: An oil phase was prepared bydissolving 600 mg of bendamustine C₁₂ ester in 5 mL of dichloromethane.The oil phase was emulsified with an aqueous phase comprised of 60 mLdeionized (“DI”) water, 2.4 g HSA (lyophilized solid from Sigma-Aldrich,St. Louis, Mo.) and 6.6 g mannitol using an IKA Ultra-Turrex hand heldhomogenizer to obtain a coarse emulsion. This emulsion was passed fivetimes through a Microfluidics M-110P high pressure homogenizer at about30,000 psi. The dichloromethane was removed from the resultingnanoparticle suspension using a rotory-evaporator and the resultingaqueous suspension was diluted to bring the total volume to 100 mL withDI water. This suspension was then portioned in 10 mL aliquots into 30mL serum vials and lyophilized.

Bendamustine C₁₂ Ester HSA Nanoparticles with poly-Lactic glycolic Acid(PLGA): An oil phase was prepared by dissolving 300 mg of bendamustineC₁₂ ester and 500 mg of PLGA (50/50 lactic to glycolic with a MW of7,000-17,000; Aldrich Part# 719897) in 2.5 mL of dichloromethane. Theoil phase was emulsified with an aqueous phase comprised of 30 mL DIwater, 1.2 g HSA (lyophilized solid from Sigma-Aldrich) and 3.3 gmannitol using and IKA Ultra-Turrex hand held homogenizer to obtain acoarse emulsion. This emulsion was passed five times through aMicrofluidics M-110P high pressure homogenizer at about 30,000 psi. Thedichloromethane was removed from the resulting nanoparticle using aroto-evaporator and the resulting aqueous suspension was diluted tobring the total volume to 50 mL with DI water. The resultingnanoparticles had a particle size of 90.5 nm (Z_(avg)) as measured byMalvern Zetasizer. This suspension was then portioned in 10 mL aliquotsinto 30 mL serum vials and lyophilized.

Preparation of PEGylated Nanoparticle Formulations of BendamustineEsters of the Invention: A series of nanoparticle formulations wereprepared with a PEG coating, which was reported in the literature(Alexis, F., Molecular Pharmaceutics, 5, (2008), 505-515) to provide a“stealth” coating and aid in the particle ability to avoid the body'simmune system. The incorporation of PEG was done by using a PEG basedsurfactant (copolymer of PEG and poly lactic acid) instead of HSA orusing bioconjugate chemistry to covalently attach PEG groups to the freeNH₂ groups on the surface of the HSA nanoparticles. Both systems showedincreased plasma circulation times in PK studies.

Bendamustine C₁₂ Ester HSA Nanoparticles with Polyethyleneglycol (PEG)Coating: An oil phase was prepared by dissolving 600 mg of bendamustineC₁₂ ester in 5 mL of dichloromethane. The oil phase was emulsified withan aqueous phase comprised of 60 mL DI water, 2.4 g HSA (lyophilizedsolid from Sigma-Aldrich) and 6.6 g mannitol using and IKA Ultra-Turrexhand held homogenizer to obtain a coarse emulsion. This emulsion waspassed five times through a Microfluidics M-110P high pressurehomogenizer at 30,000 psi. The dichloromethane was removed from theresulting nanoparticle suspension using a roto-evaporator and theresulting aqueous suspension was diluted to bring the total volume to 50mL with DI water. The suspension of nanoparticles were then diluted into200 mL of a 100 mM pH 8.5 borate buffer and the particle size andzeta-potential of the resulting nanoparticles were measured using aMalvern Zetasizer. The nanoparticles had a particle size of 78.9 nm(Z_(avg)) and a surface charge of −13.0 mV. The suspension was stirredand 150 mg of methoxy-PEG_(5,000)-n-hydroxysuccinimide ester (LaysanPolymer) was added. The reaction was mixed for ˜90 minutes at roomtemperature and the particle size and zeta-potential were re-measured.The nanoparticles particle size was found to be 83.6 nm (Zavg) and thezeta-potential was −7.35 mV. The nanoparticles were buffer exchanged andconcentrated with a 6.6% (wt/wt) mannitol solution and a 50,000 MWCOdiafiltration cartridge. This suspension was then portioned in 10 mLaliquots into 30 mL serum vials and lyophilized.

Bendamustine C₁₂ Ester Nanoparticles with poly-Lactic glycolic Acid(PLGA) and polyoxyEthylene Lactic Acid copolymer (PELA) surfactant: Anoil phase was prepared by dissolving 300 mg of bendamustine C₁₂ esterand 500 mg of PLGA (50/50 lactic to glycolic with a MW of 7,000-17,000;Aldrich Part# 719897) in 2.5 mL of dichloromethane. The oil phase wasemulsified with an aqueous phase comprised of 30 mL DI water, 0.6 g acopolymer of PEG-5,000-poly lactic acid 1,000 (copolymer prepared usingprocedure from A. Lucke, Biomaterials, 21, (2000), 2361-2370) and 3.3 gmannitol using and IKA Ultra-Turrex hand held homogenizer to obtain acoarse emulsion. This emulsion was processed for 5 minutes through aMicrofluidics M-110P high pressure homogenizer at 30,000 PSI. Thedichloromethane was removed from the resulting nanoparticle suspensionusing a roto-evaporator and the resulting aqueous suspension was dilutedto bring the total volume to 50 mL with DI water. The resultingnanoparticles had a particle size of 248.0 nm (Z_(avg)) as measured byMalvern Zetasizer. This suspension was then portioned in 10 mL aliquotsinto 30 mL serum vials and lyophilized.

Bendamustine C₁₂ Ester HSA Nanoparticles from Concentrate: An oil phasewas prepared by dissolving 4.8 g of Bendamustine C₁₂ ester in 13.4 g ofdichloromethane. The oil phase was emulsified with an aqueous phasecomprised of 111 g deionized (“DI”) water and 9 g HSA using an IKAUltra-Turrex hand held homogenizer to obtain a coarse emulsion. Thisemulsion was passed five times through a Microfluidics M-110P highpressure homogenizer at about 30,000 psi. The resulting nanoemulsionconcentrate was stabilized by adding 12 g NaCl and mixing untildissolved. Stabilization can also be achieved using other methods knownin the art, for example, other salts, controlled heating, and/or pHadjustments. Cross-linking with, for example, glutaraldehyde, can alsoassist in preventing aggregation.

The dichloromethane was removed from the resulting nanoparticlesuspension using a rotory-evaporator. The resulting aqueous suspensionwas mixed with 12 g of sucrose and DI water was added to bring the totalweight to 480 g. This suspension was then portioned in 7.5 mL aliquotsinto 20 mL serum vials and lyophilized.

Solutions of the HSA nanoparticles exhibited a slow particle sizeincrease with time. Some solutions reached ˜200 nm in size within 12-24hours as measured by dynamic light scattering. The nature of thisincrease was investigated using c-TEM in order to determine if thenanoparticles were aggregating or if excess protein in the formulationwas adding on to the surface of the particle to increase the particlesize. A vial of lyophilized nanoparticles was reconstituted and imagedusing c-TEM (FIG. 21). The same vial of nanoparticles was allowed tostand at room temperature for 24 hours then re-imaged (FIG. 22). Theoriginal sample contained spherical particles with a size distributionbetween 25-60 nm. The aged sample exhibited an increase in sizedistribution to 35-130 nm with no sign of particle aggregation. Bothsamples contain a population of 1-3 nm particles which are consistentwith “free” protein. These results suggest that “free” HSA is adding tothe surface of the nanoparticles while in solution to cause a slowincrease in particle size. Several strategies may be employed tomitigate this process including change in pH, change in osmolarity,solvent addition and diafiltration to remove excess protein.

Protocol for Preparing Tumor Cell Isolates: Charles River Labs athymicnude mice bearing MDA-MB-231 breast carcinoma cell line subcutaneousxenografts (5×10⁶ cells in matrigel) were sacrificed to make a tumorisolate. Using sterile instruments and working aseptically, the tumorswere removed from the euthanized animal. The tumor was placed in a 50 mLsterile conical tube and 5 mL of trypsin was added. The tumor was cutinto small pieces and incubated at 37° C. for 30 minutes. Afterincubation, a cell strainer was placed on a second, sterile, 50 mLconical tube and the contents of the first tube were placed in the cellstrainer. The tissue was forced through the filter using the flat end ofa syringe plunger. The cell strainer was washed with 5 mLs of mediacontaining FBS. The cells were spun down and the supernatant wasdiscarded. The cells were resuspended in 20 mLs of complete mediacontaining P/S and placed in a 75 cm flask. This data is set forth inTable 1.

General Procedure for MTS Cell Assay: Human carcinoma cells (2,000cells/well) were incubated with the desired concentration of the testmolecule (0-200 μM) for 72 hours. The cells were then incubated with MTSsolution (Promega) for 1-2 hours, and the compounds effects on cellproliferation was determined by measuring the absorbance at 490 nm. Cellgrowth was expressed as a percent of the appropriate control (placebo).This data was used to calculate an IC₅₀ for each compound. All datareported was the mean±SE of three independent experiments. This data isset forth in Table 1.

TABLE 1

MB-231 (HBC) H-460 (NSCLC) R₁ IC₅₀ (μM) R² IC₅₀ (μM) R² —OH 13-160.88-0.95 4-26 0.83-0.95 —CH₃ 66 0.95 1.2 0.9  —C₄H₉ 13.3  0.91 15.730.9  —C₆H₁₃ 43.7  0.93 7.4 0.89 —C₈H₁₇ 53.1  0.86 49 0.89 —C₁₀H₂₁ 38.890.87 23.76 0.93 —C₁₂H₂₅ 33.3  0.78 28.8  0.69 —C₁₄H₂₉ >200 0.83 129.9 0.87 —C₁₅H₃₁ >200 0.93 >200 0.66 —C₁₆H₃₃ >200 0.85 77.8  0.9  —C₁₈H₃₇N.D. N.D. 29 R² = coefficient of determination

The foregoing biological data is also depicted in FIG. 1. Plasma levelsof bendamustine after administration of the foregoing esters aredepicted in FIG. 20.

Tumor Efficacy

Procedure for Nude Mouse MDA-MB-231 Tumor Efficacy Study: Charles RiverLabs athymic nude mice were subcutaneous injected with 5×10⁶ human tumorcells in matrigel. The tumor volume was monitored until an average tumorsize of ˜150 mm³ was obtained in the mouse population The mice were thenrandomized in to one of the nine treatment groups (summarized in tablebelow) with a population of 10 mice per group (n=10). Each of theformulations was dosed at 100 μL fixed volume via tail vein injection onday 1 and day 2 of the study. Mice were weighed and the tumor volumemeasured every 3-4 days for 3 weeks of the study duration. This data isdepicted in Table 2 and FIG. 2.

TABLE 2 Summary of Tumor Efficacy Data Against MB-231 of Bendamustine(BM1), Bendamustine C₁₂ Ester, and Bendamustine C₁₄ Ester: % TumorInhibition, % Morbidity/Mortality, and Tumor and Plasma Levels. Levelsat 1 hour (ng/mL) % Tumor Ester BM1 mg/kg Inh. % Morbidity/MortalityTumor Plasma Tumor Plasma Solution BM1 37.5 77 10/0 — — 558 906 C₁₂ 5584 40/0 425 0 1932 4110 C₁₂ 80 94  80/20 644 16 4400 11650 C₁₄ 80 9040/0 396 14 7 21 HSA C₁₂ 55 79 10/0 261 2 1845 6307 nanoparticle C₁₂ 8091  70/40 762 292 7320 18757 C₁₂ 100 97 100/70 1565 39 15050 23933 C₁₄80 86.5 60/0 10430 5727 5500 15110

Procedure for Nude Mouse H460 Tumor Efficacy Study: H460 tumor cells(large cell lung cancer) were cultured in RPMI-1640 medium containing10% FBS and with 95% air and 5% CO₂ at 37° C. When reaching to 80-90%confluent, the cells were detached by 0.25% Trypsin-EDTA solution within5-10 minutes, neutralized with fresh cultured medium, and counted by acell counter (Cellometer, Auto T4 by Nexcelom). 2×10⁶ cells/100 ul inthe mix of medium and Matrigel (1:1 ratio) solution was injected intoright back flank of each nu/nu mouse. The implanted mice were monitoredand measured with electric calipers. The study started when the tumorsreached ˜150 mm³ in size. The mice were measured and randomized into 9groups with 10 mice in each group per the below table:

Tumor Efficacy Dosing Groups Dose (mg/kg Free Compound Formulation BaseEq.) Vehicle Control Solution NA Bendamustine HCl TREANDA 37.5Bendamustine C₁₂ ester Solution 55 Bendamustine C₁₂ ester Solution 80Bendamustine C₁₂ ester Nanoparticle 55 Bendamustine C₁₂ esterNanoparticle 80 Bendamustine C₁₂ ester Nanoparticle 100 Bendamustine C₁₄ester Solution 80 Bendamustine C₁₄ ester Nanoparticle 80

The formulations were administrated as 100 μL dose volume through tailvein injection within 30 minutes after compounds were formulated. Allthe mice were weighed and tumors measured twice weekly. At the last dayof the study, plasma, tumor, lung, liver, spleen, left kidney, brain andlegs were collected and quickly frozen for further analysis two hourspost-dosing. The results are shown in Table 3.

TABLE 3 Summary of Tumor Efficacy Data Against H-460 of Bendamustine(BM1), Bendamustine C₁₂ Ester, and Bendamustine C₁₄ Ester: % TumorInhibition, % Morbidity/Mortality, and Tumor and Plasma Levels. Levelsat 1 hour (ng/mL) % Tumor Ester BM1 mg/kg Inh. % Morbidity/MortalityTumor Plasma Tumor Plasma Solution BM1 37.5 41.5  0/0 — — 1472 1550 C₁₂55 66  20/20 163 12 3760 6943 C₁₂ 80 75 100/50 206 12 5180 13600 C₁₄ 8081 100/40 8570 32233 4650 24633 HSA C₁₂ 55 47 10/0 154 0 1761 4743nanoparticle C₁₂ 80 64 40/0 321 0 2997 8053 C₁₂ 100 70 50/0 437 2 356512386 C₁₄ 80 40 10/0 4570 3648 1872 4467

Pharmacokinetic Study Experimentals

Procedure for Nude Mouse Tumor Efficacy PK Group: A portion of the tumorbearing mice were dosed as a satellite PK group. Each group was dosed asdescribed for the tumor efficacy group, however the PK group waseuthanized and tissue samples collected at 1, 3 and 6 hours post dosingon day 2. The tissues collected included blood, lung, liver and tumor.Samples were analyzed for bendamustine HCl and the corresponding esteranalogue as described in the LC-MS protocol section.

LC-MS Experimental Protocol for PK Studies: Plasma and other tissueswere prepared for high performance liquid chromatography (HPLC)/massspectrometric analysis according to a standard protocol followingprotein precipitation with acetonitrile containing an internal standard.The samples were then analyzed for both bendamustine HCl andbendamustine esters of the invention and alprenolol (internal standard)via HPLC coupled with tandem mass spectrometry. Tissue samples werehomogenized in sodium phosphate buffer and the value obtained from theassay was multiplied by 3 to correct for dilution during processing.

Animal Dosing Protocol for PK studies: Adult animals (Charles River,Kingston, N.Y.; n=3 or 4/time point) were used in all experiments. Themice or rats were not fasted overnight prior to IV dose administrationvia the lateral tail vein. IV doses were administered in a fixed dosevolume of 100 μL in mice or a dose volume of 1 mL/kg in rats. The micewere sacrificed by decapitation and trunk blood was collected intoheparinized tubes at the sampling times stipulated. For bloodcollection, each rat (unanesthetized) was placed in a clear Plexiglas®restraining tube, and blood samples (approximately 0.25 mL) were drawnfrom a lateral tail vein into heparinized collection tubes at thesampling times stipulated. (Note: No pre-dose samples were obtained.)The exception to this procedure was the last sampling time in which therats were sacrificed by decapitation and trunk blood was obtained ratherthan blood via a tail vein. The blood samples were placed on wet iceuntil centrifuged to separate plasma. The plasma fraction wastransferred into clean, dry tubes, frozen on dry ice and stored atapproximately −20° C. pending analysis. Whole brains and other highlyperfused organs (liver, lung, spleen, kidney and heart) were rapidlyremoved at the predetermined time points and frozen on dry ice. Alltissue samples were also stored at approximately −20° C. pendinganalysis.

Pharmacokinetic Analysis: The plasma concentration data for all mice andrats were entered into Excel spreadsheets in preparation forpharmacokinetic analysis. Mean pharmacokinetic parameters were estimatedby non-compartmental analysis (Gibaldi and Perrier 1982) of the plasmaconcentration versus time data using WinNonlin software (ProfessionalVersion 4.1, Pharsight Corporation, Palo Alto, Calif.). The terminalrate constant for elimination from plasma (β) was estimated by linearregression of the terminal portion of the semi-logarithmic plasmaconcentration versus time curve. The apparent terminal half-life (t1/2)was calculated as 0.693 divided by β. The area under the plasmaconcentration versus time curve from time zero to the time of the lastmeasurable concentration (AUC0-t) after a single dose was determined bythe linear trapezoidal rule. The area from zero to infinity (AUC0-∞) wascalculated as the sum of AUC0-t and the area extrapolated from the lastmeasurable concentration to infinity (Clast/β). Concentrations pre-dosewere all assumed to be zero for the purpose of calculation of the AUC.Any concentration that was below the limit of quantification (BLQ) afterthe last quantifiable sampling time was considered to be an empty valuefor the purpose of calculation of the AUC; it was treated as zero forthe calculation of the mean concentration for a given sampling time.

Data from the pharmacokinetic studies is depicted in Tables 4-17.

TABLE 4 Liquid Formulation Nanoparticle Formulation Plasma BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 5.7 ND 6.0 ND AUC_(0-t),66885 ND 55296 ND ng * h/mL AUC_(0-∞), 67131 ND 55417 ND ng * h/mL

The data from Table 4 is also depicted in FIG. 8.

TABLE 5 Liquid Formulation Nanoparticle Formulation Blood BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 6.3 ND 5.7 ND AUC_(0-t),91549 4424 58021 951 ng * h/mL AUC_(0-∞), 91856 ND 58112 ND ng * h/mL

The data from Table 5 is also depicted in FIG. 9.

TABLE 6 Liquid Formulation Nanoparticle Formulation Brain BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h ND ND ND ND AUC_(0-t), 9284299 1010 149 ng * h/mL AUC_(0-∞), ND ND ND ND ng * h/mL

The data from Table 6 is also depicted in FIG. 10.

TABLE 7 Liquid Formulation Nanoparticle Formulation C₁₂ C₁₂ LiverBendamustine Ester Bendamustine Ester t_(1/2), h 4.2 0.8 2.6 4.5AUC_(0-t), ng * h/mL 13901 2564 43785 13355 AUC_(0-∞), ng * h/mL 141112586 44713 13586

The data from Table 7 is also depicted in FIG. 11.

TABLE 8 Liquid Formulation Nanoparticle Formulation Lung BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 2.0 7.6 5.1 8.5 AUC_(0-t),22229 12619 15075 28327 ng * h/mL AUC_(0-∞), 22286 13246 15590 32297ng * h/mL

The data from Table 8 is also depicted in FIG. 12.

TABLE 9 Liquid Formulation Nanoparticle Formulation Spleen BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 2.3 3.1 1.6 5.4 AUC_(0-t),10201 25874 2598 13111 ng * h/mL AUC_(0-∞), 10362 25927 2735 13578 ng *h/mL

The data from Table 9 is also depicted in FIG. 13.

TABLE 10 Liquid Formulation Nanoparticle Formulation Kidney BendamustineC₁₂ Ester Bendamustine C₁₂ Ester t_(1/2), h 6.4 5.3 7.0 4.9 AUC_(0-t),19489 2383 9665 1766 ng * h/mL AUC_(0-∞), 19966 2605 10725 1802 ng *h/mL

The data from Table 10 is also depicted in FIG. 14.

TABLE 11 Plasma, Blood, and Organ Levels of Bendamustine in Rat AfterAdministration of Bendamustine C₁₂ Ester Liquid Formulation, 30 mg/mL, 1mL/kg FIG. 15 Plasma Blood Brain Liver Lung Spleen Kidney t_(1/2), h 5.76.3 ND 4.2 2.0 2.3 6.4 AUC_(0-t), ng * h/mL 66885 91549 9284 13901 2222910201 19489 AUC_(0-∞), ng * h/mL 67131 91856 ND 14111 22286 10362 19966

The data from Table 11 is also depicted in FIG. 15.

TABLE 12 Plasma, Blood, and Organ Levels of Bendamustine C₁₂ Ester AfterAdministration of Bendamustine C₁₂ Ester Liquid Formulation, 30 mg/mL, 1mL/kg FIG.16 Plas- ma Blood Brain Liver Lung Spleen Kidney t_(1/2), h NDND ND 0.8 7.6 3.1 5.3 AUC_(0-t), ND 4424 299 2564 12619 25874 2383 ng *h/mL AUC_(0-∞), ND ND ND 2586 13246 25927 2605 ng * h/mL

The data from Table 12 is also depicted in FIG. 16.

TABLE 13 Plasma, Blood, and Organ Levels of Bendamustine in Rat AfterAdministration of Bendamustine C₁₂ Ester Nanoparticle Formulation, 30mg/mL, 1 mL/kg FIG. 17 Plasma Blood Brain Liver Lung Spleen Kidneyt_(1/2), h 6.0 5.7 ND 2.6 5.1 1.6 7.0 AUC_(0-t), ng * h/mL 55296 580211010 43785 15075 2598 9665 AUC_(0-∞), ng * h/mL 55417 58112 ND 4471315590 2735 10725

The data from Table 13 is also depicted in FIG. 17.

TABLE 14 Plasma, Blood, and Organ Levels of Bendamustine C₁₂ Ester AfterAdministration of Bendamustine C₁₂ Ester Nanoparticle Formulation, 30mg/mL, 1 mL/kg FIG. 18 Plas- ma Blood Brain Liver Lung Spleen Kidneyt_(1/2), h ND ND ND 4.5 8.5 5.4 4.9 AUC_(0-t), ND 951 149 13355 2832713111 1766 ng * h/mL AUC_(0-∞), ND ND ND 13586 32297 13578 1802 ng *h/mL

The data from Table 14 is also depicted in FIG. 18.

TABLE 15 Plasma Levels of Bendamustine in Rat After Dosing BendamustineC₁₂ Ester Nanoparticles at 3 mg-eq/kg, i.v.: Comparison of DifferentFormulations FIG. 19 Formulation Treanda HSA HSA/PLGA PLGA/PELA HSAw/PEG t_(1/2), h 0.16 ± 0.01 0.63 ± 0.09 0.41 ± 0.04  2.1 ± 0.3 1.6 ±0.3 AUC_(0-t), ng * h/mL 1609 ± 77  773 ± 58  1120 ± 113  856 ± 93 1194± 186  AUC_(0-∞), ng * h/mL 1631 ± 81  781 ± 58  1132 ± 115  959 ± 921250 ± 203  Vd, L/kg 0.42 ± 0.04 3.6 ± 0.7 1.6 ± 0.2 10.0 ± 2.2 6.1 ±1.8 CL, mL/min/kg 31 ± 2  65 ± 5  46 ± 4  54 ± 5 44 ± 7  Mean ± SD, n =4 Bendamustine C₁₂ Ester Nanoparticles

The data from Table 15 is also depicted in FIG. 5.

TABLE 16 Plasma Levels of Bendamustine C₁₂ Ester in Rat After DosingBendamustine C₁₂ Ester Nanoparticles at 3 mg-eq/kg, i.v.: Comparison ofDifferent Formulations FIG. 20 Formulation HSA HSA/PLGA PLGA/PELA HSAw/PEG t_(1/2), h ND 0.12 ± 0.01  2.3 ± 0.0  0.20 ± 0.01 AUC_(0-t), ng *h/mL 21 ± 2 18 ± 5  17 ± 3 55 ± 4 AUC_(0-∞), ng * h/mL ND 21 ± 7  36 ± 556 ± 4 Vd, L/kg ND 47 ± 15 428 ± 59 23.3 ± 2.5 CL, mL/min/kg ND 4194 ±1071 2109 ± 252 1335 ± 99  Mean ± SD, n = 4

The data from Table 16 is also depicted in FIG. 6.

TABLE 17 Plasma Levels of Bendamustine C₁₆ Ester, Cyclohexyl Ester, and5-Decanyl Ester in Rat After Dosing Solution Formulations at 3 mg-eq/kgi.v. Bendamustine Ester Bendamustine Ester Bendamustine Ester Plasma C16bendamustine ester 5-decanyl ester Cy-hexyl bendamustine ester t_(1/2),h 0.46 0.28 0.16 0.20 AUC_(0-t), 505 1208 633 504 ng * h/mL AUC_(0-∞),617 1214 642 All < MQL 517 All < MQL ng * h/mL Vd, L/kg 3.3 1.0 1.1 1.6CL, 82 41 78 96 mL/min/kg 1.85% solvent 1.6% solvent 1.4% solvent Mean,n = 3 Diluted into saline from 1/1/1 DMA/PG/Solutol

Another embodiment of the invention, bendamustine C₁₄ ester wasformulated into a nanoparticle intraveneous formulation according to themethods described above and administered to CD-1 mice. The amount ofbendamustine (BM1) and bendamustine C₁₄ ester was determined in the miceplasma. The results of these experiments are summarized in Table 18.

TABLE 18 Plasma BM1 C₁₄ Ester BM1 C₁₄ Ester BM1 C₁₄ Ester 30 mg-eq/kg 55mg-eq/kg 80 mg-eq/kg t_(1/2), h 1.17 1.52 0.92 0.98 0.95 1.09 AUC₀₋₆,ng * h/mL 4507 31693 4152 48934 6007 46335 AUC_(0-∞), ng * h/mL 452431741 4188 49020 6068 46485 Vd, L/kg ND 3.2 ND 2.5 ND 5.2 CL, mL/min/kgND 24 ND 29 ND 56 Mean, n = 3 Albumin Nanoparticle

PEG-ylated esters of bendamustine were also tested. Data for PEG-2000and PEG-5000 esters of bendamustine is depicted in Tables 19 and 20below. This data is also depicted in FIG. 19.

TABLE 19 Plasma Levels of Bendamustine in Rat Dased as BendamustinePEG-2000 Ester, 3 mg-eq/kg i.v., 1 mL/kg Bendamustine Rat 1 Rat 2 Rat 3Rat 4 Mean st. dev. sem t_(1/2), h 0.46 0.23 0.50 0.22 0.36 0.15 0.07AUC₀₋₆, ng * h/mL 2100 2866 1494 1379 1960 682 341 AUC_(0-∞), ng * h/mL2109 2868 1506 1382 1966 680 340 Vd, L/kg 0.95 0.35 1.46 0.70 0.87 0.460.23 CL, mL/min/kg 24 17 33 36 28 9 4 bendamustine 3 mg-eq/kg, 1 mL/kgPEG-2000 ester

TABLE 20 Plasma Levels of Bendamustine in Rat Dased as BendamustinePEG-5000 Ester, 3 mg-eq/kg i.v., 1 mL/kg Bendamustine Rat 1 Rat 2 Rat 3Rat 4 Mean st. dev. sem t_(1/2), h 0.16 0.48 0.47 0.48 0.40 0.16 0.08AUC₀₋₆, ng * h/mL 808 1735 1572 1050 1291 435 217 AUC_(0-∞), ng * h/mL817 1741 1579 1058 1299 434 217 Vd, L/kg 0.84 1.19 1.29 1.99 1.32 0.480.24 CL, mL/min/kg 61 29 32 48 42 15 8 bendamustine 3 mg-eq/kg, 1 mL/kgPEG-5000 ester

Analysis of the In-Vitro Stability of Bendamustine Esters

Tumor S9 preparation: Charles River Labs athymic nude mice bearingbreast (MB-231) or non-small-cell lung cancers (H460) were sacrificed.Tumors were immediately removed and rinsed with ice-cold 1.15% KCl. Thetumors were weighed, cut and minced. Minced tissues were mixed with 4×(v/w) ice-cold SET buffer (250 mM sucrose, 5.4 mM Na₂EDTA and 20 mMTris, pH 7.4) and homogenized with tissue homogenizers. Homogenates weretransferred into clean polycarbonate ultracentrifuge tubes and spun at10,000 g at 4° C. for 20 min. Lipid at the top of the ultracentrifugetubes was removed with cotton swabs, and the supernatant (S9) aliquotswere stored in a −80° C. freezer.

In vitro incubation: Incubation mixture containing 50 mM phosphatebuffer (pH 7.4), an NADPH- (reduced nicotinamide adenosine diphosphate)regenerating system and 1 mg/mL tumor S9 were pre-warmed in a 37° C.water bath. Reactions were initiated by adding 1 μL of bendamustine C6,C8, C12 or C14 ester into separate incubation mixtures to obtain finalconcentrations of each bendamustine ester of 10 μM. At designed timepoints, 100-μL aliquots of the incubation mixtures were removed andmixed with 400 μL of stop solution (4 or 8 μM tiagabine [IS] in asolution of 0.1% formic acid/acetonitrile). All samples werevortex-mixed and placed on ice for at least 10 min and then the proteinwas precipitated by centrifuging in an Eppendorf 5417R centrifuge at14000 rpm×8 min. The supernatant was transferred into HPLC vials and 10μL was injected for analysis using high performance liquidchromatography with tandem mass spectrometric detection.

LC-MS/MS method: The LC-MS/MS system consisted of a Shimadzu HPLC and aSciex API 4000 MS. The chromatography was performed on a Phenomenex00B-4448-B0, Luna PFP(2) column (50×2 mm, 5 μm particle size). The totalmobile phase flow rate was 0.5 mL/min. The gradient began at 70% mobilephase A (0.1% aqueous trifluoroacetic acid) and 30% mobile phase B (100%acetonitrile). The proportion of mobile phase B was then linearlyincreased to 95% within 0.5 min and was maintained at that ratio for 1.3min, re-equilibrating to initial conditions within 1 min. The massspectrometer was tuned to the respective optimal conditions for eachbendamustine ester, monitoring transitions of 442.2/340.1 (C6),470.2/340.1 (C8), 526.3/340.1 (C12) and 554.3/340.1 (C14).

Data from the in-vitro stability studies is set forth in FIGS. 3 and 4.

Electron Microscopy Experimentals

Sample Preparation for c-TEM Study: Sample was solubilized by adding 7.8mL of water for injection (WFI) to the sample and mixed by inverting byhand. Sample dissolved quickly, ˜2-5 minutes, with no visibleundissolved particles in the solution. The sample was preserved invitrified ice supported by carbon coated holey carbon films on 400 meshcopper grids. The sample was prepared by applying a 3 μL drop ofundiluted sample solution to a cleaned grid, blotting away with filterpaper and immediately proceeding with vitrification in liquid ethane.Grids were stored under liquid Nitrogen until transferred to theelectron microscope for imaging.

c-TEM Imaging Parameters: Electron microscopy was performed using an FEITecnai T12 electron microscope, operating at 120KeV equipped with an FEIEagle 4K×4K CCD camera. The grid was transferred into the electronmicroscope using a cryostage that maintains grids at a temperature below−170 C. Images of the grid were acquired at multiple scales to assessthe overall distribution of the specimen. After identifying potentiallysuitable target areas for imaging at lower magnifications, highmagnification images were acquired at nominal magnifications of 52,000×(0.21 nm/pixel), and 21,000× (0.50 nm/pixel). The images were acquiredat a nominal underfocus of −4 μm (52,000×) and −5 μm (21,000×) andelectron doses of ˜10-15 e/Å2.

Results of the electron microscopy experiments is depicted in FIG. 7.

Cross-Linking Experiments with HSA Nanoparticles Formulation ofBendamustine Esters

Circulation times of bendamustine and bendamustine esters of theinvention can be extended using HSA-based nanoparticle formulationwherein the protein moieties are covalently cross-linked after thenanoparticle structures are formed. See, e.g., K. Langer et al.International Journal of Pharmaceutics 347 (2008) 109-117. This wouldprovide more structure to the surface coating and would prevent a rapidrelease of the nanoparticle contents. This could also provide “stealth”protection of the nanoparticle by introducing a PEG group to thecross-linking agent. Effective encapsulation and hardening of the HSAnanoparticle was demonstrated using the commercially availabledialdehyde, glutaraldehyde. Introduction of an appropriately-sized PEGmoiety could be added using, for example, the trifunctional PEGcross-linking agent prepared as shown below:

Procedure: HSA nanoparticles were diluted with DI water to aconcentration of 1.5 mg/mL C14 ester of bendamustine, which correspondsto a 6 mg/mL concentration of HSA. The resulting suspension ofnanoparticles was then aliquoted in 1 mL portion into five glass vialsoutfitted with a magnetic stir bar. The appropriate amount of a 50%glutaraldehyde solution was added and each vial was capped and stirredat room temperature over-night. Each sample was then diluted 1 to 10into N-methylpyrrolidone (NMP) and the sample spun for ˜2 minutes usinga micro-centrifuge to remove HSA and cross-linked HSA nanoparticles. Thesupernatant was then analyzed by HPLC and the concentration (peak area)of the C14 ester of bendamustine was determined to confirm particleencapsulation. The table below shows the concentration ofun-encapsulated C14 ester of bendamustine as a function of the μL ofgluturaldehyde:

μL of glutaraldehyde HPLC Peak Area 0 4882.02 2 4788.99 5 4778.11 104714.02 20 29.55

The data shows that the addition of glutaraldehyde at a ratio of 3.33μL/mg of HSA was suitable to result in a system of cross-linked,bendamustine ester-containing nanoparticles

In Vivo Multiple Myeloma Model Materials and Methods

RPMI 8226 (Human Plasmacytoma, Myeloma. B Cells) ATCC #CCL-155;

ECM Gel (Matrigel), Sigma-Aldrich, Cat #E1270, 5 ml

RPMI (Beit Haemek, Lot: 1110235)

Velcade® (Bortezomib) 3.5 mg lyophilized in vial, Lot# BIZSC00

Bendamustine (Lot #TD-D0815, API Lot #00039P0012)

Bendamustine C12 ester nanoparticles (C12NP), Lot #2861-242-22, 17.6mg/vial

Sodium chloride

Water for injections (DEMO S.A.)

Test Animals

80 CB.17 SCID female mice, 4-6 weeks old, 16-20 grams, obtained fromHarlan animal breeding center

Cells Preparation

Cells (originated from ATCC) were cultured on RPMI medium. Cellsuspension was centrifuged and resuspended in 50% Matrigel/HBSS to afinal concentration of 7×10⁷ cells/ml. The suspension was implanted s.c.in the right flank of the anesthetized mouse at a volume of 100 μl.

Compounds Preparation

VELCADE® was prepared once a week. Seven ml saline were added to theoriginal vial containing 3.5 mg powder resulting in 0.5 mg/ml. Three mlof this solution were added to 27 ml saline to receive 0.05 mg/mlconcentration.

bendamustine preparation: 13.5 mg was dissolved in 3.6 ml of 1:1 mixtureof 0.9% saline/5% mannitol just before the treatment. 1.05 ml of thissolution was added to 0.95 ml diluent for 2 mg/ml solution.

C12NP preparation: 3.1 ml SWFI was added into sample vial containing17.62 mg just before the treatment. 1.05 ml of this solution was addedto 0.95 ml diluent for 2 mg/ml solution.

Experimental Design

Mice were implanted subcutaneously, with 7×106 RPMI 8226 cells/mouse (in50% Martigel/HBSS) on Day 0. On day 21, mice were sorted by the optimalaverage tumor volume (˜150 mm³) and were allocated into eight groups of9 mice each.

Gr. N Agent Route Dose & schedule 1 9 WFI iv Days 1 & 2 2 9 Velcade iv0.5 mg/kg biweekly 3 9 Bendamustine iv 20 mg/kg on days 1&2 4 9Bendamustine iv 37.5 mg/kg on days 1&2 5 9 C12NP iv 20 mg/kg on days 1&26 9 C12NP iv 37.5 mg/kg on days 1&2

It should be noted that the nature of SCID mice (i.e., severelyimmune-compromised) make them more fragile/sensitive animals, and theyare therefore less able to tolerate the doses of C12NP used in NUDE mice(which are comparatively less immune-compromised). Specifically, dosingSCID mice at 100 mg/kg and 70 mg/kg revealed unacceptable toleranceissues (i.e., more than 20% body weight loss)(data not shown). It isbelieved that this (tolerance difference between nude vs. SCID) is astrain-related phenomenon. As such, the study proceeding using doses of37.5 mg/kg and 20 mg/kg of C12NP.

Statistical Analysis

Tumor volume was calculated as follows:

$\pi \times \left( \frac{width}{2} \right)^{2} \times {{length}.}$

The analysis of weight gain and tumor volume progression was made usingone-way ANOVA followed by Tukey post-hoc comparisons.

Results

The treatment responses are summarized in FIGS. 23 and 24. All thetreatments except C12NP 20 mg/kg significantly inhibited tumor growthcompared to control group (see data in Table 21). At 37.5 mg/kg, theefficacy of Bendamustine and C12NP were similar, with 81% and 70%inhibition of tumor growth. However, as can be seen in FIG. 24, thetolerability of C12NP was better than Bendamustine as measured by lessweight loss in animals treated with C12NP.

TABLE 21 Summary of Results (day 46) Δ mean tumor Max BW volume, No. ofNo. of (mean) No. of No. og Compound Regimen (mean ± se) % TGI PR CRreduction TRD nTRD 1. Vehicle WFI iv on 597 ± 71 days 1 and 2 2.Velcade ® i.v. biwk 226 ± 35 62*** 0 0 −0.4% 0 0 0.5 mg/kg day 20 3.Bendamustine iv on days 1 205 ± 44 66*** 0 0 −2.7% 0 0 20 mg/kg and 2day 22 4. Bendamustine iv on days 1 113 ± 23 81*** 0 0 −6.2% 0 0 37.5mg/kg and 2 day 25 5. C12NP 20 mg/kg iv on days 1 408 ± 49 32 0 0 −1.5%0 0 and 2 day 25 6. C12NP 37.5 mg/kg iv on days 1 180 ± 31 70*** 0 0−3.1% 0 0 and 2 day 25 ***p < 0.001 (one-way Anova, Tukey post-hoc test)

1. (canceled)
 2. A method of treating cancer in a patient comprisingadministering to said patient a pharmaceutical composition, wherein saidpharmaceutical composition comprises: (a) a compound of formula (I)

wherein R₁ is C₈-C₂₄ alkyl, or a pharmaceutically acceptable saltthereof; and (b) a means for increasing the circulation time of thecompound of formula (I), or a pharmaceutically acceptable salt thereof,in an aqueous environment.
 3. The method according to claim 2, whereinsaid cancer is in the form of a solid tumor or a liquid tumor.
 4. Themethod according to claim 3, wherein said cancer is in the form of asolid tumor.
 5. The method according to claim 4, wherein said cancer issarcoma, bladder cancer, cervical cancer, testicular cancer, melanoma,glioblastoma, colon cancer, head and neck cancer, ovarian cancer,prostate cancer, breast cancer, or lung cancer.
 6. The method accordingto claim 2, wherein said cancer is in the form of a liquid tumor.
 7. Themethod according to claim 6, wherein said cancer is lymphocyticleukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma,or acute lymphocytic leukemia.
 8. The method according to claim 2,wherein in the compound of formula (I) R₁ is C₁₀-C₂₄ alkyl.
 9. Themethod according to claim 2, wherein in the compound of formula (I) R₁is C₁₂-C₂₄ alkyl.
 10. The method according to claim 2, wherein in thecompound of formula (I) R₁ is C₁₄ ⁻C₂₄ alkyl.
 11. The method accordingto claim 2, wherein in the compound of formula (I) R₁ is C₁₀ alkyl. 12.The method according to claim 2, wherein in the compound of formula (I)R₁ is C₁₂ alkyl
 13. The method according to claim 2, wherein in thecompound of formula (I) R₁ is C₁₄ alkyl.
 14. The method according toclaim 2, wherein in the compound of formula (I) R₁ is C₁₅ alkyl.
 15. Themethod according to claim 2, wherein in the compound of formula (I) R₁is C₁₆ alkyl.
 16. A method of treating cancer in a patient comprisingadministering to said patient a pharmaceutical composition, wherein saidpharmaceutical composition comprises: (a) a compound of the formula

or a pharmaceutically acceptable salt thereof; and (b) a means forincreasing the circulation time of the compound, or a pharmaceuticallyacceptable salt thereof, in an aqueous environment.
 17. The methodaccording to claim 16, wherein said cancer is in the form of a solidtumor or a liquid tumor.
 18. The method according to claim 17, whereinsaid cancer is in the form of a solid tumor.
 19. The method according toclaim 18, wherein said cancer is sarcoma, bladder cancer, cervicalcancer, testicular cancer, melanoma, glioblastoma, colon cancer, headand neck cancer, ovarian cancer, prostate cancer, breast cancer, or lungcancer.
 20. The method according to claim 17, wherein said cancer is inthe form of a liquid tumor.
 21. The method according to claim 20,wherein said cancer is lymphocytic leukemia, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma, or acute lymphocytic leukemia.